Methods and compositions for treating autoimmune diseases

ABSTRACT

The invention features methods for increasing or maintaining the number of functional cells of a predetermined type, for example, insulin producing cells of the pancreas, blood cells, spleen cells, brain cells, heart cells, vascular tissue cells, cells of the bile duct, or skin cells, in a mammal (e.g., a human patient) that has injured or damaged cells of the predetermined type.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/632,452 (now U.S. Pat. No. 8,173,129), filed Dec. 7, 2009, which is adivisional of, and claims priority from U.S. application Ser. No.10/358,664 (now U.S. Pat. No. 7,628,988), filed Feb. 5, 2003, whichclaims the benefit of the filing date of U.S. Provisional ApplicationNo. 60/392,687, filed Jun. 27, 2002, each of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The invention relates to repairing and regenerating damaged tissue in ahuman. Such damage may result from an existing autoimmune disease, ormay be the result of a non-autoimmune insult. We have previously shownthat eliminating autoimmune cells and re-educating the immune system areimportant components of an effective treatment of an autoimmune disease(described in U.S. patent application Ser. Nos. 09/521,064, 09/768,769,and Ryu et al., Journal of Clinical Investigations, “Reversal ofEstablished Autoimmune Diabetes by Restoration of Endogenous Beta CellFunction,” 108:31-33, 2001), which are hereby incorporated byreference). While an autoimmune disease may be successfully treated, theindividual may nonetheless have significant tissue damage as a result ofthe prior autoimmune attack.

Many tissues have an innate ability to repair themselves once the damagecausing insult is eliminated, but this ability to repair damagedecreases in correlation with the duration of the insult. For example,the regenerative capacity of endogenous pancreatic islets is virtuallyeliminated in long-term Type I diabetics, i.e., patients who have hadthe disease for more than 15 years. In cases where the endogenous tissuehas lost its regenerative capacity, the damage may be repaired byproviding exogenous tissue to the individual, for example, a transplant.A promising treatment for diabetes, islet transplantation, has been thesubject of human clinical trials for over ten years. While there havebeen many successes with islet transplantation in animals, these haveoccurred where the animals are diabetic due to chemical treatment,rather than natural disease. The only substantiated peer reviewedstudies using non-barrier and non-toxic methods and showing success withislet transplants in naturally diabetic mice use isogeneic (self)islets. The isogeneic islets were transplanted into non-obese diabetic(NOD) mice with active diabetes, which were pre-treated with TNF-alpha(tumor necrosis factor-alpha); BCG (Bacillus Clamette-Guerin, anattenuated strain of mycobacterium bovis); or CFA (Complete Freund'sAdjuvant), which is an inducer of TNF-alpha (Rabinovitch et al., J.Immunol. 159:6298-6303, 1997). This approach is not clinicallyapplicable primarily because syngeneic islets are not available.Furthermore, existing cell replacement strategies have not preventedend-stage diseases or permanently reversed insulitis. In the allograftsetting of islet transplantation, grafts are eventually rejected, evenwith immunosuppression. Furthermore, diabetic host treatments such asbody irradiation and bone marrow transplantation are unacceptably toxic,rendering the short-term alternative of insulin therapy more attractive.

Recently, islet transplantation has achieved limited success in clinicaltrials, such as that observed for allogenic transplants combined withmulti-drug immunosuppression therapy, with type 1 diabetic patientshaving a sustained return to normoglycemia over a 6 month period. Theseresults have been obtained with continuous, and sometimes toxic, drugtherapy, often in the setting of a simultaneous life-saving renaltransplant. However, these moderately successful islet transplants showfailures after about one year, speculated to be due in part to the drugtherapy itself inducing insulin resistance. The earlier failure of islettransplants in type 1 diabetics, compared to non-diabetic patientsreceiving islet transplants (such as in cancer patients who have hadtheir pancreas removed), raises the concern that immunosuppressivetherapy shows greater efficacy for graft rejection over autoimmunityprevention. Lending credence to these concerns is the observation of theinefficiency of immunosuppression therapy for the prevention of graftrejection of allogenic or xenogeneic islet transplants in animal studiesusing non-obese diabetic (NOD) mice.

We previously described a transplantation method to introduce allogeneicand xenogeneic tissues into non-immunosuppressed hosts in which thecells are modified such that the donor antigens are disguised from thehost's immune system (U.S. Pat. No. 5,283,058, which is herebyincorporated by reference). Generally, masked islets or transgenicislets with ablated MHC class I molecules are only partially protectedfrom recurrent autoimmunity in NOD mice (Markmann et al.,Transplantation 54:1085-1089, 1992). A need exists for methods ofregenerating damaged tissue that are not only applicable to tissuedamage that results from autoimmune attack, but also to non-autoimmuneinduced damage.

SUMMARY OF THE INVENTION

The invention features methods for organ regeneration in a mammal (e.g.,a human patient). Accordingly, in a first aspect, the invention featuresa method for increasing or maintaining the number of functional cells ofa predetermined type in a mammal (i) who has injured or damaged cells ofthe predetermined type or who has a deficiency of cells of thepredetermined type (e.g., a mammal with a lower than normal number ofthese cells or a mammal lacking these cells) and (ii) who does not havean autoimmune disease. This method involves administering to the mammala composition that induces lymphopenia and that increases the number ofcells of the predetermined cell type in the animal. In desirableembodiments, the composition activates a receptor on the surface ofcells of the predetermined cell type or on the surface of precursorcells that differentiate into cells of the predetermined cell type inthe mammal. Desirably, the method also includes administering cells ofthe predetermined cell type to the mammal. In desirable embodiments, themethod includes administering cells that recapitulate a developmentalsequence (e.g., endoderm with mesoderm; endoderm with ectoderm; orectoderm with mesoderm to promote the regeneration of the tissue ofinterest). In some embodiments, the method also includes administeringprecursor cells that differentiate into cells of the predetermined celltype to the mammal. In particular embodiments, the method also includesinducing damage to the cells of a predetermined type in the mammal orinducing damage in a site of the mammal in which cells of thepredetermined type are desirable. For example, damage can be induced incells of the predetermined type or cells of another type within 10inches, 5 inches, 1 inch, 10 cm, 5 cm, 1 cm, 10 mm, or 1 mm of thelocation in which cells of the predetermined type are desirable.

In another aspect, the invention features a method for treating orstabilizing an established autoimmune disease in a mammal. This methodinvolves (a) administering to the mammal a first composition thatselectively kills a predetermined subpopulation of blood cells, in anamount sufficient to selectively kill at least 10%, preferably at least75%, of the subpopulation of blood cells in the mammal, (b) repeatingstep (a) one or more times, and (c) optionally monitoring the glucoselevel in the mammal two or more times. Desirably, the method alsoincludes administering to the mammal a second composition thatselectively kills a predetermined subpopulation of blood cells, in anamount sufficient to selectively kill at least 10%, preferably at least75%, of the subpopulation of blood cells in the mammal. In someembodiments, the method includes determining whether the mammal has asubpopulation of blood cells with higher than normal sensitivity to thefirst composition prior to step (a).

In a related aspect, the invention features another method for treatingor stabilizing an established autoimmune disease in a mammal. Thismethod involves (a) administering to the mammal a first composition thatselectively kills a predetermined subpopulation of blood cells, in anamount sufficient to selectively kill at least 10%, preferably at least75%, of the subpopulation of blood cells in the mammal, (b) repeatingstep (a) one or more times, and (c) optionally maintaining the bloodglucose level in the mammal within a normal range. Desirably, the methodalso includes administering to the mammal a second composition thatselectively kills a predetermined subpopulation of blood cells, in anamount sufficient to selectively kill at least 10%, preferably at least75%, of the subpopulation of blood cells in the mammal. In someembodiments, the method includes determining whether the mammal has asubpopulation of blood cells with higher than normal sensitivity to thefirst composition prior to step (a).

In another aspect, the invention features a method for treating,stabilizing, or preventing an autoimmune disease (e.g., an establishedautoimmune disease) and/or increasing or maintaining the number offunctional cells of a predetermined type in a mammal. This methodinvolves (a) administering to the mammal a composition that selectivelykills a predetermined subpopulation of blood cells, in an amountsufficient to selectively kill at least 10%, preferably at least 75%, ofthe subpopulation of blood cells in the mammal, and (b) prior to, after,or concurrently with step (a), administering to the mammal cells thathave the potential to differentiate into the predetermined type or thatare of the predetermined cell type. In one embodiment, the methodincludes determining, prior to step (a), whether the blood of the mammalcontains a subpopulation of blood cells with higher than normalsensitivity to the composition to be administered; if this is the case,the decision to employ the composition is reinforced.

In another aspect, the invention features a method for treating,stabilizing, or preventing an autoimmune disease (e.g., an establishedautoimmune disease) and/or increasing or maintaining the number offunctional cells of a predetermined type in a mammal. This methodinvolves (a) administering to the mammal a first composition thatselectively kills a pre-determined subpopulation of stimulated bloodcells, in an amount sufficient to selectively kill at least 10%,preferably at least 75%, of the subpopulation of stimulated blood cellsin the mammal, (b) prior to, after, or concurrently with step (a),administering to the mammal a second composition that selectively killsa predetermined subpopulation of unstimulated blood cells, in an amountsufficient to selectively kill at least 10%, preferably at least 75%, ofthe subpopulation of unstimulated blood cells in the mammal, and (c)prior to, after, or concurrently with steps (a) or (b), administering tothe mammal cells that have the potential to differentiate into thepredetermined type or that are of the predetermined cell type. In oneembodiment, prior to step (a), the method includes determining whetherthe blood of the mammal contains a subpopulation of stimulated bloodcells with increased sensitivity to the first composition; if this isthe case, the decision to employ the first composition is reinforced. Inanother embodiment, prior to step (b), the method includes determiningwhether the blood of the mammal contains a subpopulation of unstimulatedblood cells with increased sensitivity to the second composition; ifthis is the case, the decision to employ the second composition isreinforced.

In another aspect, the invention features a method for treating,stabilizing, or preventing an autoimmune disease (e.g., an establishedautoimmune disease). This method involves administering to the mammal acomposition that selectively kills a pre-determined subpopulation ofunstimulated blood cells, in an amount sufficient to selectively kill atleast 10%, preferably at least 75%, of the subpopulation of unstimulatedblood cells in the mammal. In one embodiment, the method includesdetermining whether the blood of the mammal contains a subpopulation ofunstimulated blood cells with increased sensitivity to the compositionto be administered; if this is the case, the decision to employ thecomposition is reinforced.

In another aspect, the invention features a method for treating,stabilizing, or preventing an autoimmune disease (e.g., an establishedautoimmune disease). This method involves administering to the mammal acomposition that selectively kills a pre-determined subpopulation ofstimulated blood cells, in an amount sufficient to selectively kill atleast 10%, preferably at least 75%, of the subpopulation of stimulatedblood cells in the mammal. In one embodiment, the method includesdetermining whether the blood of the mammal contains a subpopulation ofstimulated blood cells with increased sensitivity to the composition tobe administered; if this is the case, the decision to employ thecomposition is reinforced.

In another aspect, the invention features a method for treating,stabilizing, or preventing an autoimmune disease (e.g., an establishedautoimmune disease). This method involves administering to the mammal acomposition that selectively kills a pre-determined subpopulation ofblood cells, in an amount sufficient to selectively kill at least 10%,preferably at least 75%, of the subpopulation of blood cells in themammal. In one embodiment, the method includes determining whether theblood of the mammal contains a subpopulation of blood cells withincreased sensitivity to the composition to be administered; if this isthe case, the decision to employ the composition is reinforced.

In another aspect, the invention features a method of increasing ormaintaining the number of functional cells of a predetermined type in amammal. This method involves (a) administering to the mammal acomposition that selectively kills a predetermined subpopulation ofstimulated blood cells, in an amount sufficient to selectively kill atleast 10%, preferably at least 75%, of the subpopulation of stimulatedblood cells in the mammal, and (b) prior to, after, or concurrently withstep (a), administering to the mammal cells that have the potential todifferentiate into the predetermined type or that are of thepredetermined cell type. In one embodiment, prior to step (a), themethod includes determining whether the blood of the mammal contains asubpopulation of stimulated blood cells with increased sensitivity tothe composition to be administered; if this is the case, the decision toemploy the composition is reinforced. In some embodiments, the mammalhas an injured or diseased organ that has increased normal functionalactivity after administration of the composition.

In another aspect, the invention features a method of increasing ormaintaining the number of functional cells of a predetermined type in amammal. This method involves (a) administering to the mammal acomposition that selectively kills a predetermined subpopulation ofstimulated blood cells, in an amount sufficient to selectively kill atleast 10%, preferably at least 75%, of the subpopulation of stimulatedblood cells in the mammal, and (b) prior to, after, or concurrently withstep (a), administering to the mammal one or more precursor cells thatdifferentiate into cells of the predetermined type in vivo. Desirably,the precursor cells are stem cells and step (a) is performed prior tostep (b). In other embodiments, non-islet cells are administered for thetreatment or prevention of diabetes. In certain embodiments, the methodalso involves regulating blood sugar levels in diabetic patients using,e.g., a glucose clamp or administered insulin. Desirably, a compositionthat kills unstimulated blood cells is administered to the patient in anamount sufficient to selectively kill a subpopulation of unstimulatedblood cells in the patient.

In another aspect, the invention features a method for increasing ormaintaining the number of functional cells of a predetermined type in amammal. This method involves (a) administering to the mammal one or morecells of blood origin or endothelial origin, and (b) prior to, after, orconcurrently with step (a), administering to the mammal a compositionthat selectively kills a predetermined subpopulation of stimulated bloodcells, in an amount sufficient to selectively kill at least 10%,preferably at least 75%, of the subpopulation of stimulated blood cellsin the mammal. In some embodiments, the patient has an autoimmunedisease (e.g., diabetes) or an increased risk for an autoimmune disease.Desirably, a composition that kills unstimulated blood cells isadministered to the patient in an amount sufficient to selectively killa subpopulation of unstimulated blood cells in the patient.

In another aspect, the invention features a method for increasing ormaintaining the number of functional cells of a predetermined type in amammal. This method involves (a) administering to the mammal one or morecells of blood origin or of endothelial, mesoderm or ectodermic origin,and (b) prior to, after, or concurrently with step (a), administering tothe mammal a composition that promotes or recapitulates the embryonicprogram of cellular differentiation in the host. In desirableembodiments, the method includes administering to the mammal aproteasome activity-promoting substance, such as gamma interferon. Insome embodiments, the method includes administering to the mammal anagent that increases Flk or Flt expression or function. Examples includeTNF-, IL-1, HAT, or NF-B induction, or cAMP inhibition, using agentsknown to achieve these functions. Other examples include stimulation ofAP-2, EGF-1, Sp1, AP-1, NFkB, GATA stimulation with the induction ofPECAM-1, activator protein-2, CT-rich Sp1 binging activity, PDGF-A,PDGF-B, monocyte chemoattractant protein-1, TF, Ets1, SCL/Tal-1, FGF,HATs P/CAF, CBP/p300 and HIF-2alpha (HRF, EPAS, HLF). These functionsmay also be achieved by TGF-beta inhibition, TGF-beta receptor blockade,or inhibition of CREB (camp response element binding protein). Incertain embodiments, the method includes administering to the mammal anagent that increases VEGF, VEGF1, VEGF2, VEGF1R, or VEGF2R expression orfunction, such as a VEGF polypeptide or a nucleic acid molecule encodinga VEGF polypeptide or substance that activates the promoter of a VEGFprotein receptor. VEGF polypeptides include full-length VEGF proteins,as well as biologically active VEFG fragments. These agents are in somecases preferred for mesoderm/endodermal activation for differentiation.For mural differentiation (cells usually of neural crest or pericardialorigins), host treatment with PDGF or PDGF-BB can desirably be includedin the method. For BV endothelium differentiation or regrowth of tissue,treatment of the host with an FGF and/or IGF-1 can be desirable.Furthermore, for promotion of regeneration can in some instances beaccomplished using just one agent, or with two or more agents,administered with or without pluripotent cells.

In another aspect, the invention features a method of increasing ormaintaining the number of functional cells of a predetermined type in amammal. This method involves administering to the mammal a compositionthat selectively kills a predetermined subpopulation of stimulated bloodcells, in an amount sufficient to selectively kill at least 10%,preferably at least 75%, of the subpopulation of stimulated blood cellsin the mammal. In one embodiment the method includes determining whetherthe blood of the mammal contains a subpopulation of stimulated bloodcells with higher than normal sensitivity to the composition to beadministered; if this is the case, the decision to employ thecomposition is reinforced. In some embodiments, the mammal has aninjured or diseased organ that increased normal functional activityafter administration of the composition.

In another aspect the invention features a method of increasing ormaintaining the number of functional cells of a predetermined type in amammal with an autoimmune disease or an increased risk for an autoimmunedisease. This method involves administering to the mammal one or moreprecursor cells that differentiate into one or more cells of thepredetermined type in vivo or that promote proliferation of endogenouscells of the predetermined type in vivo. The differentiated cell(s) willeventually present MHC class I and peptide, and the MHC class I has atleast one allele that matches an MHC class I allele expressed by themammal.

In another aspect, the invention features a method of increasing ormaintaining the number of functional cells of a predetermined type in amammal. This method involves (a) administering to the mammal one or morecells of the predetermined type, and (b) prior to, after, orconcurrently with step (a) administering to the mammal a compositionthat kills unstimulated blood cells, in an amount sufficient toselectively kill at least 10%, preferably at least 75%, of thesubpopulation of unstimulated blood cells in the mammal. In oneembodiment, prior to step (a), the method includes determining whetherthe blood of the mammal contains a subpopulation of unstimulated bloodcells with higher than normal sensitivity to the composition to beadministered. In some embodiments, the mammal has an injured or diseasedorgan that has increased normal functional activity after administrationof the composition.

In another aspect, the invention features a method of increasing ormaintaining the number of functional cells of a predetermined type in amammal. This method involves administering to the mammal a compositionthat selectively kills a predetermined subpopulation of stimulated bloodcells, in an amount sufficient to selectively kill at least 10%,preferably at least 75%, of the subpopulation of stimulated blood cellsin the mammal. In one embodiment the method includes determining whetherthe mammal has a subpopulation of stimulated blood cells with higherthan normal sensitivity to the composition to be administered. In someembodiments, the mammal has an injured or diseased organ that hasincreased normal functional activity after administration of thecomposition.

In yet another aspect, the invention features a method of increasing ormaintaining the number of functional cells of a predetermined type in amammal. This method involves (a) administering to the mammal one or moreprecursor cells that differentiate into one or more cells of thepredetermined type in vivo, and (b) prior to, after, or concurrentlywith step (a), administering to the mammal a composition thatselectively kills a predetermined subpopulation of unstimulated bloodcells, in an amount sufficient to selectively kill at least 10%,preferably at least 75%, of the subpopulation of unstimulated bloodcells in the mammal. In one embodiment the method includes determiningwhether the blood of the mammal contains a subpopulation of unstimulatedblood cells with higher than normal sensitivity to the composition to beadministered. In some embodiments, the mammal has an injured or diseasedorgan that has increased normal functional activity after administrationof the composition.

In still another aspect, the invention features a method for treating,stabilizing, or preventing a disease, disorder, or condition in amammal. This method (a) administering to the mammal a first compositionthat selectively kills a predetermined subpopulation of blood cells, inan amount sufficient to selectively kill at least 10%, preferably 75%,of a first subpopulation of blood cells in the mammal, and (b) prior to,after, or concurrently with step (a), administering to the mammal asecond composition that selectively kills a predetermined subpopulationof blood cells, in an amount sufficient to selectively kill at least10%, preferably 75%, of a second subpopulation of blood cells in themammal. The first subpopulation and the second subpopulation are eitherpartially overlapping subpopulations or non-overlapping subpopulations.Desirably, first subpopulation and the second subpopulation are indifferent stages of differentiation. In some embodiments, the firstsubpopulation and the second subpopulation are in different stages ofthe cell cycle. In various embodiments, the first subpopulation and thesecond subpopulation are sensitive to different inducers of cell death.In some embodiments, the first subpopulation and the secondsubpopulation undergo cell death through different pathways. Inparticular embodiments, one subpopulation undergoes cell death throughapoptosis and the other subpopulation undergoes cell death throughnecrosis. In some embodiments, the patient has arthritis (e.g.,rheumatoid arthritis). In particular embodiments, a patient witharthritis is not administered any cells. In other embodiments, thepatient is administered chondrocytes or cells that differentiate intochondrocytes. In some embodiments, the patient has injured or damagedcells of a predetermined cell type. Desirably, the method also involvesadministering to the patient one or more cells that have the potentialto differentiate into one or more cells of the predetermined type orthat are of the predetermined cell type. The cells can be administeredprior to, after, or concurrently with the administration of the firstand/or second compositions.

In another aspect, the invention features a method for treating,stabilizing, or preventing a disease, disorder, or condition in amammal. This method involves (a) administering to the mammal a firstcomposition that selectively kills a predetermined subpopulation ofunstimulated blood cells, in an amount sufficient to selectively kill atleast 10%, preferably at least 75%, of the subpopulation of unstimulatedblood cells in the mammal, and (b) prior to, after, or concurrently withstep (a), administering to the mammal a second composition thatselectively kills a predetermined subpopulation of stimulated bloodcells, in an amount sufficient to selectively kill at least 10%,preferably at least 75%, of the subpopulation of stimulated blood cellsin the mammal. In some embodiments, the mammal has injured or damagedcells of a predetermined cell type. In certain embodiments, the methodinvolves administering to the mammal one or more cells that have thepotential to differentiate into one or more cells of the predeterminedtype or that are of the predetermined cell type.

In another aspect, the invention a method of increasing or maintainingthe number of functional cells of a predetermined type in a humanpatient. This method involves inducing damage or uncovering endogenousdamage (e.g., damage that promotes engraftment of transplanted cells) tothe cells of a predetermined type in the patient. Endogenous damage canbe measured using, e.g., blood tests, (e.g., liver function tests, testsfor glucose levels, neurologic tests, or blood cell tests), visual orradiographic tests, or functional tests. A first composition thatselectively kills a predetermined subpopulation of unstimulated bloodcells is administered to a patient, in an amount sufficient toselectively kill at least 10%, preferably at least 75%, of thesubpopulation of unstimulated blood cells in the patient. Prior to,after, or concurrently with the administration of the first composition,a second composition that kills stimulated blood cells is administeredto the patient in an amount sufficient to selectively kill asubpopulation of stimulated blood cells in the patient. Prior to, after,or concurrently with the prior steps, one or more cells that have thepotential to differentiate into one or more cells of the predeterminedtype or that are of the predetermined cell type cells are administeredto the patient.

In another aspect, the invention features a method for increasing ormaintaining the number of functional cells of a predetermined type, forexample, pancreas cells that produce insulin, brain cells, heart cells,vascular tissue cells, cells of the bile duct, chondrocytes, or skincells, in a human patient who has injured or damaged cells or adeficiency of cells of the predetermined type. This method includes (a)administering to the patient MHC class I and peptide (e.g., soluble MHCclass I and peptide or MHC class I and peptide present on the surface ofa cell); (b) prior to, after, or concurrently with step (a),administering to the patient cells that have the potential todifferentiate into the predetermined type or that are of thepredetermined type; and (c) prior to, after, or concurrently with step(b), inducing transient lymphopenia in the patient or in a blood samplefrom the patient that is re-administered to the patient. In someembodiments, steps (a) and (b) are performed concurrently byadministering cells that have the capacity to present MHC class 1 andpeptide and that have the potential to differentiate into thepredetermined type or that are of the predetermined type.

In another aspect, the invention features another method of increasingor maintaining the number of functional cells of a predetermined type ina human patient. This method involves (a) identifying endogenous damageof or inducing damage to the cells of a predetermined type in thepatient, (b) exposing the patient to MHC class I and peptide, (c) priorto, after, or concurrently with step (b), administering to the patientcells that have the potential to differentiate into the predeterminedtype or that are of the predetermined cell type, and (d) prior to,after, or concurrently with step (c), inducing transient lymphopenia inthe patient or in a blood sample from the patient that isre-administered to the patient. In some embodiments, steps (b) and (c)are performed concurrently by administering cells that have the capacityto present MHC class I and peptide and that have the potential todifferentiate into the predetermined type or that are of thepredetermined type.

In desirable embodiments of any of the aspects of the invention, themethods include administering to the mammal a cell (e.g., an endothelialcell or mesenchymal cell) that promotes proliferation of the precursorcells or cells of the predetermined cell type at the site of desiredregeneration, or a cell that can itself differentiate into thepredetermined cell type. Desirably, the methods also includeadministering a cytokine, chemokine, or growth factor to the mammal.Alternatively, the methods also can include the pretreatment ofmesenchymal or endothelial cell precursors with a cytokine, chemokine,or growth factor prior to their administration to the mammal. Exemplarycells of the predetermined type are islet cells that produce insulin,blood cells, spleen cells, chondrocytes, brain cells, heart cells,vascular tissue cells, cells of the bile duct, epithelial cells,endothelial cells, endoderm cells, mesoderm cells, mesenchymal cells,cells of mesenchymal origin, and skin cells. Desirable cells thatdifferentiate into cells of the predetermined type in vivo aresplenocytes, bone marrow derived cells, Hoechst 33342 positive cells,brain cells, CNS positive cells, hepatocytes, mesenchymal cells,mesodermal cells, endothelial cells, mural cells, and fetal cells. Insome embodiments, the cells that differentiate into cells of thepredetermined type in vivo are semi-allogeneic or isogeneic. In variousembodiments, the cells that differentiate into cells of thepredetermined type in vivo fail to express Fas or FasL. Desirable bloodcells are T-cells, B-cells, or macrophages. Mesenchymal cells that arederived from the blood, spleen, or bone marrow and defined as Hox 11⁺,CD90⁺, Flk^(low), CD34⁺ or CD45⁺ are highly desirable. Other desirablecells are cells that do not, at the time they are administered, expressMHC class I and peptide, but which have the capacity to do so in vivopost-transplantation, e.g., by stimulation with the appropriateantigens. In some embodiments, the MHC class I and peptide aresemi-allogeneic or isogeneic. In certain embodiments, the composition isa compound that crosslinks or binds to a T-cell receptor (TCR) or othersurface protein on a T-cell. In various embodiments, the composition isTNF-alpha, a TNF-alpha agonist, or a TNF-alpha inducing substance. Insome embodiments, the composition binds or activates a death receptor.Exemplary TNF-alpha inducing substances include Complete Freund'sAdjuvant (CFA), ISS-ODN, microbial adjuvants, such as cell wallcomponents with LPS-like activity, cholera particles, E. coli heatlabile enterotoxin, E. coli heat labile enterotoxin complexed withlecithin vesicles, ISCOMS-immune stimulating complexes, chemicaladjuvants, such as polyethylene glycol andpoly(N-2-(hydroxypropyl)methacrylamide), synthetic oligonucleotidescontaining CpG or CpA motifs, lipid A derivatives, such asmonophosphoryl lipid A, MPL, muramyl dipeptide derivatives, BacillusClamette-Guerin (BCG), Tissue Plasminogen Activator (TPA),lipopolysaccharide (LPS), Interleukin-1 (IL-1), Interleukin-2 (IL-2), UVlight, lymphotoxin, cachectin, a transcription factor-like nuclearregulator-2 (TNFR-2) agonist, a neutral blocking antibody to aB-lymphocyte stimulator (BLyS) receptor or soluble protein, anintracellular mediator of the TNF-alpha signaling pathway, a NFκBinducing substance, lymphotoxin, cachectin, IRF-1, STAT1, an agonist ofan ICS-2lgAS promoter element, a lymphokine, LPS, an agonist, such as anantibody, to a TNF-superfamily receptor or soluble form of aTNF-superfamily member, a combination of TNF-alpha and an anti-TNFR-1antibody, or a combination of TNF-alpha and a TNFR.

In desirable embodiments, the method includes administering to themammal a proteasome activity promoting substance, such as gammainterferon. In some embodiments, the method includes administering tothe mammal an agent that increases Flk or Flt expression or function.Examples include TNF-, IL-1, HAT, or NF-B induction, or cAMP inhibition.In certain embodiments, the method includes administering to the mammalan agent that increases VEGF, VEGF1, VEGF2, VEGF1R, or VEGF2R expressionor function, such as a VEGF polypeptide, a nucleic acid moleculeencoding a VEGF polypeptide, or a substance that activates a promoter ofthe VEGF receptor. In some embodiments, the method includesadministering to the mammal an inhibitor of Fas or FasL expression orsignaling. Desirably, the method includes maintaining the blood glucoselevel in the mammal within a normal range. In a particular embodiment,bone marrow cells or precursor (or pluripotent) cells (e.g., cord bloodcells) are administered to hasten the healing process. These cellsrecapitulate the embryonic process in adult animals by hastening thecritical mesoderm to endoderm interactions, endoderm and ectoderminteractions and mesoderm and ectoderm interactions, all of which arecrucial for organ regeneration.

In desirable embodiments of any of the various aspects of the invention,the composition that kills naïve T-cells is MHC class I and peptide, andthe MHC class I has at least one allele that matches an MHC class Iallele expressed by the patient. In some embodiments, the MHC class Iand peptide is soluble MHC class I and peptide or MHC class I andpeptide present on the surface of a cell. In some embodiments, theadministration of cells and the administration of MHC class I andpeptide to kills naïve T-cells are performed concurrently byadministering cells that have the capacity to present MHC class I andpeptide and that have the potential to differentiate into thepredetermined type or that are of the predetermined type. In someembodiments, the composition that kills pathologic T-cells (e.g., naïveT-cells) is a compound (e.g., an antibody or antibody fragment,cytokine, lymphokine, small molecule antagonist, T-cell mitogen, orco-receptor) that crosslinks a T-cell receptor (TCR) of naïve T-cells(e.g., naïve T-cells that might otherwise develop into autoimmuneT-cells or pathologic cells that die if bound by an antibody oragonist). In some embodiments, the compound that kills a subpopulationof naïve T-cells is not BCG or is not FAS. In other embodiments, thecompound that kills a subpopulation of T-cells (e.g., naïve T-cells) isBCG, FAS, or a compound that modulates a protein kinase. An exemplarycompound that selectively kills an undesired subpopulation of naïveT-cells is -CD3 antibody, a selective TCR stimulant. Examples of methodsfor the re-selection of naïve/unstimulated T cells include the directkilling of the disease-causing T cells, direct killing of themonocyte/macrophage antigen presenting cell with deficient MHC class Iand self peptide (for example, BCG), and the re-introduction of cellscorrectly presenting MHC class I and self peptide fragments.

In desirable embodiments, the mammal has an autoimmune disease or anincreased risk for an autoimmune disease. Exemplary autoimmune diseasesinclude Alopecia Areata, Ankylosing Spondylitis, AntiphospholipidSyndrome, Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia,Autoimmune Hepatitis, Behcet's Disease, Bullous Pemphigoid,Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic Fatigue ImmuneDysfunction Syndrome (CFIDS), Chronic Inflammatory DemyelinatingPolyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CRESTSyndrome, Cold Agglutinin Disease, Crohn's Disease, Discoid Lupus,Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves'Disease, Guillain-Barré, Hashimoto's Thyroiditis, Hypothyroidism,Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura(ITP), IgA Nephropathy, Insulin dependent Diabetes, Juvenile Arthritis,Lichen Planus, Lupus, Ménière's Disease, Mixed Connective TissueDisease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris,Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, PolyglandularSyndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis,Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis,Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, RheumatoidArthritis, Sarcoidosis, Scleroderma, Sjögren's Syndrome, Stiff-ManSyndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis,Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, Wegener'sGranulomatosis, and myasthenia gravis. In some embodiments, anymetabolic disorder that is due to the injury or damage of the cells of apredetermined type or due to the autoimmune disease is controlled. Insome embodiments, the mammal has an established autoimmune disease(e.g., the mammal has symptoms of the autoimmune disease). In someembodiments, the mammal does not have an established autoimmune disease.In particular embodiments, the mammal does not have cancer or AIDS.Desirably, the mammal is a human.

MHC class I and peptide can be administered either simultaneously(together or separately) or within 24 hours of each other. In someembodiments, two or more distinct MHC class I molecules that eachcontain a different allele that matches an MHC class I allele expressedby the patient are administered to the patient. In some embodiments, MHCclass I and peptide are administered in an amount sufficient to inducetolerance to the donor cells or to the cells of the predetermined type.Desirably, the number of the autoimmune cells in the patient (e.g.,B-cells that produce a self-reacting antibody or T-cells that areactivated by presented self epitopes) decreases by at least 5, 10, 20,30, 40, 50, 60, 80, 90, 95, or 100%.

In various embodiments of the above aspects, MHC class I and peptide areadministered to the patient by administering cells that express MHCclass I and peptide and that either have the potential to differentiateinto the predetermined type or are of the predetermined cell type. Inother embodiments, a population of living or dead (e.g., irradiated)cells that express MHC class I which has at least one allele thatmatches an allele expressed by the patient and which presents a peptideare administered to the patient, and another population of cells thatdiffer from the first population of cells and have the potential todifferentiate into the predetermined type or are of the predeterminedcell type are administered to the patient. This latter population ofcells may or may not present MHC class I and peptide, and, if expressed,the MHC class I may or may not contain one or more alleles that matchthat of the patient. In some embodiments, two populations of cells eachwith a different MHC class I that matches an allele expressed by thepatient are administered.

In certain embodiments, a complex of MHC class I and peptide is formedby incubating an extracellular region of MHC class I (e.g., a solubleFab fragment) with one or more peptides (e.g., peptides from a celllysate or a library of random peptides, synthetic peptides, ornaturally-occurring peptides). Because MHC class I binds peptides withhigh affinity, the soluble MHC class I fragment binds peptides insolution. In other embodiments, complexes of MHC class I and peptide arecleaved from MHC class I-expressing cells (e.g., healthy lymphocyteswith at least one MHC class I allele that matches that of the patient)using, e.g., a protease. In yet other embodiments, cells that expressMHC class I, which may not be complexed with peptide due to potentialproblems with assembly, are isolated from the patient. A fragment of theMHC class I is cleaved from the cells and incubated with one or morepeptides, and the resulting complex of MHC class I and peptide isadministered to the same patient from which the cells were obtained orto a different patient.

In some embodiments of any of the above aspects, lymphopenia is inducedby administering to the patient an agent that is nonspecific, i.e., anagent that is toxic to lymphocytes generally, rather than targeting aparticular subset of lymphocytes or lymphopenia due to new cellulardistributions. Examples of such inducers of lymphopenia are TNF-alphaand substances that induce TNF-alpha, e.g., Complete Freund's Adjuvant(“CFA”), ISS-ODN, microbial adjuvants, such as cell wall components withLPS-like activity, cholera particles, E. coli heat labile enterotoxin,E. coli heat labile enterotoxin complexed with lecithin vesicles,ISCOMS-immune stimulating complexes, chemical adjuvants, such aspolyethylene glycol and poly(N-2-(hydroxypropyl)methacrylamide),synthetic oligonucleotides containing CpG or CpA motifs, lipid Aderivatives, such as monophosphoryl lipid A, MPL, muramyl dipeptidederivatives, Bacillus Clamette-Guerin (“BCG”), other vaccinations,Tissue Plasminogen Activator (“TPA”), lipopolysaccharide (“LPS”),Interleukin-1, Interleukin-2, UV light, lymphotoxin, cachectin, a TNFR-2agonist, a NFκB inducing substance, lymphotoxin, cachectin, IRF-1,STAT1, an agonist of an ICS-2lgAS promoter element, or the combinationof TNF-alpha and a TNFR-1 antibody. These inducers of lymphopenia canspecifically kill a subpopulation of blood cells (e.g., a subpopulationof T-cells) if administering in a dose that is sufficient tospecifically kill the subpopulation but not sufficient tononspecifically kill all blood cells. For example, autoimmune patientshave subpopulations of blood cells with increased sensitivity to celldeath: thus, low dose of these compounds are required to kill thesecells. In cases in which non-specific lymphopenia is desired (e.g., suchas the treatment of mammals without an autoimmune disease) larger dosescan be administered. A nonspecific agent can also be an intracellularmediator of the TNF-alpha signaling pathway, e.g., NFκB, Jun N-terminalkinase (“JNK”), TRAILR2, FasL, TRADD, FADD, TRAF2, RIP, MAPK, kinaseactivators, a caspase, or pro-caspase. Stimulation of a signalingpathway may involve, e.g., receptors of the TNF superfamily orintracellular mediators of these pathways. Other examples of compoundsthat induce lymphopenia include compounds that bind or activate one ormore members of the TNF receptor superfamily (e.g., TNF receptor 1 or 2,Trail-RE Trail-R2, Trail-R3, Trail-R4, OPG, Rank, Fn14, DR6, Hvem,LtbetaR, DcR3, Tramp, Fas, CD40, CD30, CD27, 4-1BB, OX40, Gitr, Ngfr,BCMA, Taxi, Baff-r, EDAR, Xedar, Troy, Relt, or CD95L). Therapeuticagents can include TNF receptor superfamily cytokine agonists orcytokine agonist antibodies. Additional compounds that directly orindirectly increase TNF-alpha can be readily identified using routinescreening assays for TNF-alpha expression levels or activity. Desirably,an inducer of lymphopenia also promotes organ formation, promotesdifferentiation of donor cells (e.g., blood cells) into a desired celltype, and/or induces damage to host cells of a predetermined cell typeto facilitate incorporation of donor cells into the desired organ. Insome embodiments, transient lymphopenia is induced for a period of timesufficient to destroy at least 10, 20, 30, 40, 50, 60, 80, 90, 95, or100% of the autoimmune cells in the patient (e.g., B-cells that producea self-reacting antibody, T-cells that are activated by presented selfepitopes, or a subset of antigen presenting cells with defective antigenpresentation). In some embodiments, that agent that kills naïve T-cellsis not BCG or FAS.

In some embodiments, one or more of the following TNF super familyligands are administered to the mammal or are upregulated byadministration of another compound to the mammal: Trail(Apo2L), RANKL,TWEAK, TNF, LT, LIGHT, LT, TL1A, FASL, CD40L, CD30L, CD27L, CD27L,4-1BBL(CD137L), OX40L(CD134L), GITRL, APRIL, BAFF, EDA1, or EDA2. Incertain embodiments, one or more of the following TNFα superfamilyreceptors are activated by administration of a compound to the mammal:TRAIL-R1(DR4), TRAIL-R2(DR5), TRAIL-R3(DCR1), TRAIL-R4(DCR2), OPG, RANK,RN14, DR6, THF-R2(CD120B), TNF-R1(CD120A), FVEM, LIBETAR, DCR3,TRAMP(DR3), FAS(CD95), CD40, CD30, CD27, 4-1BB(CD137), CD134(OX40),GITR, NGFR, BCMA, TACI, BAFFR, EDAR, XEDAR, TROY, or RELT. Desirably,one or more of the following members of death cascades are activated:FASL(CD95L:CD178) with FAS (CD95), TRAIL (APO-2L) with TRAIL-R1(DR4),TRAIL(APO2L) with TRAIL R2(DR5), or TNF with TNFR1.

Desirably, one or more of the following are administered to the mammal:IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-18,INF-alpha, IFN-beta, IFN-gamma, TFG-beta, PDGF, and/or VEGF. A smallmolecule or antibody agonist of TLR1, TLR2, TLR6, TLR3, TLR4, TLR5,TLR7, and/or TLR9 is desirably administered. Exemplary TNF superfamilymembers and their receptor agonists include TRAIL-R1, TRAIL-R2,TRAIL-R3, TRAIL-R4, OPG, RANK, Fn14, DR6, TNF-R2, TNF-R1, HVEM, LtbetaR,DcR3, TRAMP, Fas, CD30, CD27, 4-1BB, OX40, GITR, NGFR, BCMA, TACI, EDAR,XEDAR, Troy, and RELT. Biologics of diverse compositions such as BCG,BLP, fibronectin Domain A, lipoarabinomannan, LPS binding protein, LPS,lipoteichoic acid, macrophage stimulatory lipopeptide 2, manosylatedphosphatidylinositol peptidoglycan, respiratory syncytial virus proteinF, and soluble tuberculosis factor may also be administered, if desired.

Any of a wide variety of cells can be administered to the patientaccording to the invention. The cells can be cells that, compared to thedesired functional cells, are relatively undifferentiated; i.e., theycan be stem cells derived, e.g., from embryonic or fetal tissue, fromadult stem cell sources, from adult tissues harboring a subset ofpluripotent cells, or from cord blood. Alternatively, the administeredcells can be relatively more differentiated cells, e.g., cells thatstain positively for the stain Hoechst 33342 which stains the nucleus ofimmature hematopoietic cells; brain-derived cells; cells derived fromnon-brain CNS tissue (e.g., spinal cord); hepatocytes; chondrocytes;splenocytes; bone marrow-derived cells; cells of blood or lymphoidorigin; or parenchymal cells. In certain embodiments, the administeredcells are not islets, not beta cells, or not insulin-producing betacells. In various embodiments, cells other than the predetermined celltype are administered to the patient and form the predetermined celltype in vivo. For example, in some embodiments, cells other than isletsare administered to the patient and form insulin-producing islets invivo. Desirably, the cells are administered to the same human from whichthey were obtained or to another human. It is also contemplated thatdonor cells from other mammals can be used. Exemplary donor mammaliancells are from pigs or primates such as monkeys.

In some embodiments, patients (e.g., patients without an autoimmunedisease) are administered cells that have been genetically engineered(e.g. by elimination of genes encoding cell death proteins such as Fas,FasL, or caspases), chemically pre-treated, or biologically pre-treated(e.g., treated with an antibody or antibody fragment reactive with Fasor FasL) to exhibit reduced resistance to spontaneous cell death. Incertain embodiments, a compound that decreases the expression level oractivity of Fas or FasL is administered to the donor cells or to thehost. In some embodiments, the cells are allogeneic, semi-allogeneic, orisogeneic. Exemplary MHC class I-expressing cells that can be used inthe invention include monocytes, macrophages, dendritic cells, B-cells,Langerhans cells, epithelial cells, mesenchymal cells, and parenchymalcells. Other cells express MHC class I at lower levels and can also beused in the present methods. Desirably, at least 20, 40, 60, 80, 90, 95,or 100% of the administered cells express MHC class I complexed withpeptide. In some embodiments, cells present MHC class 1 and peptidebefore and after they are administered to the patient. In otherembodiments, cells present MHC class 1 and peptide only after they areadministered to the patient. In some cases, the cells differentiate invivo into cells that present MHC class 1 and peptide.

In some embodiments, one or more death receptors (e.g., the deathreceptors listed in FIG. 5) are inactivated on the donor cells or one ormore intracellular signaling proteins that mediate cell death areinactivated in the donor cell to prevent death of the transplantedcells. For example, FLIP can be used to down regulate Fas/FasLexpression. In other embodiments, extracellular inhibition or reductionin IL2 (e.g., inhibition due to chemicals or antibodies) is used toupregulate FLIP which then down regulates FAS. In other embodiments, thedonor cells have a blockage of IL2R, such as the binding of a chemical(e.g., a non-lytic antibody fragment) to IL2R to inhibit binding of IL2to IL2R and thus IL2-mediated upregulation of FAS. In other embodiments,one or more members of the intracellular pathway for FAS activation areinhibited in the donor cells prior to transfer. Examples include theinhibition of the translation of transcription factors such as cFOS,cJAN, PKC, Lck, Zap70, MAPK, Itk (IL-2 inducible T cell kinase) and JNK.In particular embodiments, the transcription or translation oftranscription factors is transiently inhibited with antisenseoligonucleotides or by RNA interference (RNAi).

The number of functional cells maintained according to the invention canbe increased by further treating the patient with a substance thatincreases proteasome activity; this treatment can specifically increasethe immuno-proteasome subunits LMP2, LMP7, and LMP10, rendering thecells or host more responsive to the growth promoting activities of VEGFpathways or NF B pathways, which are important for cellularregeneration. Administration of the proteasome-enhancing substance canbe carried out at any time during the method of the invention; e.g.,prior to or following the administration of the cells and/or MHC class Iand peptide; or prior to or following induction of transientlymphopenia. The proteasome activity-increasing substance can be, e.g.,gamma interferon; VEGF or a substance that increases VEGF level oractivity, such as a nucleic acid molecule encoding VEGF or an activefragment thereof; fetal liver kinase-1/kinase domain region (Flk-1/KDR)or a substance that increases Flk level or activity; or fms-liketyrosine kinase-1 (Flt-1) or a substance that increases Flt level oractivity. Additional proteasome activity-increasing substances includecompounds that inhibit the expression or signaling of Fas or FasL (e.g.,an anti-Fas or anti-FasL antibody) and/or promote the viability ofendogenous or exogenously administered pluripotent cells.

Desirably, the number of cells of the desired cell type that are presentat least one day, one week, one month, or one year after treatment usingthe methods of the invention increases by at least 20, 30, 50, 75, 100,200, 500, or even 1000% relative to the number of cells of that celltype that are present in the patient before treatment or the number ofcells present in a control subject (e.g., a subject who received avehicle control or a placebo). In some embodiments, the number of cellsof the desired cell type in a treated patient is at least 50, 60, 70,80, 90, or 100% of the amount of the corresponding cells in a healthypatient without a deficiency in those cells. In certain embodiments,cells of donor origin are present in the patient at least one day, oneweek, one month, or one year after treatment. For example, diabeticpatients that are treated using the present methods desirably containcells of donor origin in their pancreas after treatment.

In some embodiments, the methods of the invention are used to treatdamage or deficiency of cells in an organ, muscle, or other bodystructure or to form an organ, muscle, or other body structure.Desirable organs include the bladder, brain, nervous system tissue,blood vessels, skin, eye structures, gut, bone, muscle, ligament,esophagus, fallopian tube, heart, pancreas, intestines, gallbladder,kidney, liver, lung, ovaries, prostate, spinal cord, spleen, stomach,testes, thymus, thyroid, trachea, ureter, stomach, urethra, and uterus.For these applications, donor differentiated cells, such as cells fromany of these organs, or undifferentiated cells, such as embryonic oradult stem cells, are administered to a patient. In a desirableembodiment, a pancreas is regenerated, or the organelle representing theislets of Langerhans reappears. In some embodiments, the organ, muscle,or other body structure that is repaired or replaced was damaged, atleast in part, due to the aging process. In some embodiments, at least20, 40, 60, 80, 90, 95, or 100% of the cells of the regenerated organ(e.g., the bile ducts, endocrine portions of the pancreas, or the entirepancreas), muscle, or other body structure are of donor origin. Incertain embodiments, the precursor cells are provided in a female bypregnancy.

The methods of the invention can be used to treat, prevent, or stabilizeautoimmune diseases and diseases or conditions other than autoimmunediseases, such as diseases or injuries associated with damage to aparticular class of cells. For example, these method may be used totreat, prevent, or stabilize autoimmune diseases including, but notlimited to, Insulin dependent Diabetes, lupus, Sjogren's disease,rheumatoid arthritis, pemphigus vulgaris, multiple sclerosis,hypothyroidism, graves disease, psoriasis, premature ovarian failure(POF), and myasthenia gravis. Other examples of autoimmune diseases aredescribed herein. In these procedures, the cells that are attacked bythe recipient's own immune system may be replaced by transplanted cellsthat either are the desired cell type or that differentiate into thedesired cell type in vivo. For the treatment of type I or type IIdiabetes, insulin-producing islet cells (e.g., islet cells expressingMHC class I that has at least one allele that matches the patient andthat presents a peptide) or cells that differentiate intoinsulin-producing islet cells in vivo (e.g., cells originating fromsplenocytes, bone marrow origin cells, blood origin cells, orfibroblasts) can be transplanted into the patient. Desirably, thepatient's glucose level decreases to less than 200 mg/dl, 150 mg/dl, or120 mg/dl (in order of increasing preference).

Examples of other medical applications for these methods include theadministration of neuronal cells or cells that differentiate intoneuronal cells to an appropriate area in the human nervous system totreat, prevent, or stabilize a neurological disease such as Alzheimer'sdisease, Parkinson's disease, Huntington's disease, or ALS; or a spinalcord injury. For example, degenerating or injured neuronal cells may bereplaced by transplanted cells. Undifferentiated donor cells may beadministered, e.g., systemically or locally.

In preferred embodiments for the treatment of conditions other thanautoimmune conditions, a compound that inhibits the destruction of stemcells by an administered inducer of lymphopenia (e.g., TNF-alpha) isdesirably administered to the patient. Examples of such compoundsinclude anti-Fas antibodies. In some embodiments, stem cells or cellsthat form stem cells in vivo are administered to the patient after theadministration of the inducer of lymphopenia or lymphoid redistribution(e.g., after the destruction of endogenous autoimmune cells, such asafter 7-14 days of lymphopenia).

With respect to the therapeutic methods of the invention, it is notintended that the administration of one or more compounds (e.g.,purified or unpurified donor cells, MHC class I and peptide, and aninducer of lymphopenia) to a patient be limited to a particular mode ofadministration, dosage, or frequency of dosing; the present inventioncontemplates all modes of administration, including intramuscular,intravenous, intraperitoneal, intravesicular, intraarticular,intralesional, subcutaneous, or any other route sufficient to provide adose adequate to increase the number of desired cells. The compound(s)may be administered to the patient in a single dose or multiple doses.When multiple doses are administered, the doses may be separated fromone another by, for example, one day, one week, one month, or one year.For example, a compound that induces lymphopenia may be administeredonce a week for, e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks.It is to be understood that, for any particular subject, specific dosageregimes should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions. For example, the dosage of donorcells can be increased if more cells of a particular cell type areneeded, e.g., if the glucose levels of a diabetic patient have notreturned to normal or if an ongoing process decreases the number oractivity of cells of the predetermined cell type. The dosage of acompound that induces lymphopenia can also be increased if autoimmunecells remain in the patient, for example, if a blood sample from thepatient contains autoantibodies or contains cells with increasedsensitivity to TNF-alpha, indicating that autoimmune cells are stillpresent in the patient. Conversely, the dosage of donor cells orlymphopenia-inducing compounds can be decreased if a desired number ofcells are present in the patient or if autoimmune cells are no longerpresent. If desired, conventional treatments may be used in combinationwith the therapies of the present invention. For example, diet andexercise can be used to facilitate the control of glucose levels indiabetic patients.

Other embodiments of the present methods are disclosed in U.S. patentapplication Ser. No. 09/521,064, filed Mar. 8, 2000, and 09/768,769,filed Jan. 23, 2001, and PCT publication WO00/53209, published Sep. 14,2000, which are incorporated by reference).

DEFINITIONS

By “treating, stabilizing, or preventing a disease, disorder, orcondition” is meant preventing or delaying an initial or subsequentoccurrence of a disease, disorder, or condition; increasing thedisease-free survival time between the disappearance of a condition andits reoccurrence; stabilizing or reducing an adverse symptom associatedwith a condition; reducing the severity of a disease symptom; slowingthe rate of the progression of a disease; or inhibiting or stabilizingthe progression of a condition. Desirably, at least 10, 20, 30, 40, 60,80, 90, or 95% of the treated subjects have a complete remission inwhich all evidence of the disease disappears. In another preferredembodiment, the length of time a patient remains free of diseasesymptoms after being diagnosed with a condition and treated with atherapy of the invention is at least 10, 20, 40, 60, 80, 100, 200, oreven 500% greater than (i) the average amount of time an untreatedpatient remains free of disease symptoms or (ii) the average amount oftime a patient treated with another therapy remains free of diseasesymptoms. Desirably, the number of disease-causing white blood cellsdecreases by at least 10, 20, 30, 40, 60, 80, 95, or 100%.

By “autoimmune disease” is meant a disease in which an immune systemresponse is generated against self epitopes. Some examples of autoimmunediseases include Alopecia Areata, Ankylosing Spondylitis,Antiphospholipid Syndrome, Autoimmune Addison's Disease, AutoimmuneHemolytic Anemia, Autoimmune Hepatitis, Behcet's Disease, BullousPemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic FatigueImmune Dysfunction Syndrome (CFIDS), Chronic Inflammatory DemyelinatingPolyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CRESTSyndrome, Cold Agglutinin Disease, Crohn's Disease, Discoid Lupus,Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves'Disease, Guillain-Barré, Hashimoto's Thyroiditis, Hypothyroidism,Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura(ITP), IgA Nephropathy, Insulin dependent Diabetes, Juvenile Arthritis,Lichen Planus, Lupus, Ménière's Disease, Mixed Connective TissueDisease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris,Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, PolyglandularSyndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis,Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis,Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, RheumatoidArthritis, Sarcoidosis, Scleroderma, Sjögren's Syndrome, Stiff-ManSyndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis,Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, Wegener'sGranulomatosis, and myasthenia gravis.

“MHC class I and peptide” is commonly understood to refer to theMHC/peptide complex as it is naturally presented on the surface of acell in connection with the normal functioning of the immune system.Cytoplasmic antigens are processed into peptides by cytoplasmicproteases and the proteasome, a multicatalytic proteinase complexassociated with the Lmp2, Lmp7, and Lmp10 protein. As used herein, theterm “MHC class I and peptide” includes such naturally occurringcomplexes, and in addition includes peptides that differ from nativeantigen-derived peptides but which are nonetheless able to form acomplex with class I that is effective to maintain functional cellsaccording to the invention. Exemplary peptides that differ from nativeantigen-derived peptides may contain unnatural amino acids, e.g.,D-amino acids, as well as naturally-occurring amino acids. Preferred MHCclass I and peptide complexes are those in which a chain of amino acidsbetween 8 and 10 residues in length is correctly complexed with an MHCclass I molecule that is either semi-allogeneic, i.e., at least one MHCclass I allele is mismatched and at least one MHC class I allele ismatched between donor and recipient, or syngeneic, i.e., all MHC class Ialleles are matched between donor and recipient, where the MHC class Iand peptide complex contributes to the re-education or re-selection ofthe immune system.

In some embodiments, the MHC class I and peptide are present on thesurface of cells that are administered to the patient. Other MHC classI/peptide complexes are soluble complexes that are not expressed on thesurface of a cell. In particular embodiments, the extracellular regionof MHC class I (e.g., a Fab fragment of MHC class I) or soluble,full-length MHC class I is incubated with one or more peptides accordingto known methods under conditions that allow a peptide to bind the MHCclass I fragment, and the resulting MHC class I and peptide complex isadministered to the patient. In other embodiments, a mixture of MHCclass I and peptide are administered to the patient, and the MHC class Iand peptide bind in vivo after administration to the patient. In someembodiments, the administered MHC class I has 1, 2, 3, or 4 alleles withat least 60, 70, 80, 90, 95, or 100% sequence identity to that MHC classI expressed by the patient. Sequence identity is typically measuredusing sequence analysis software with the default parameters specifiedtherein (e.g., Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705). This software program matchessimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications.

By “functional cell,” is meant cells that carry out their normal in vivoactivity. In certain desirable embodiments of the invention, the cellsare capable of expressing endogenous self-peptide in the context of MHCclass I.

By “predetermined type,” when used in reference to functional cells, ismeant a defined cell type. For example, one skilled in the art maydecide to carry out the method of the present invention in order toincrease or maintain the number of functional islet cells in thepancreas. In this example, the cells of a predetermined type are isletcells or islet precursor cells.

Standard assays can be used to determine whether administered cells formcells of the predetermined cell type in vivo. For example, cells may beanalyzed for expression of particular proteins (e.g., proteins specificfor the predetermined cell type) using standard Western orimmunofluorescence analysis or for the expression of particular mRNAmolecules (e.g., mRNA molecules specific for the predetermined celltype) using a cDNA array (Ausubel et al., supra). Examples of othercharacteristics of the administered cells that may be analyzed todetermine whether they have been converted into the desired cell typeinclude the size of the cell, cell morphology, volume of cytoplasm, andcell function (e.g., production of insulin or other hormones).

By “semi-allogeneic,” is meant a match of at least one marker, forexample, an MHC class allele, between cells of the same type fromdifferent individuals of the same species. Desirably at least two orthree MHC class I alleles match between the donor and the host. Standardmethods may be used to determine whether an MHC class I allele expressedby a donor cell matches an MHC class I allele expressed by therecipient. For example, antibodies specific for a particular MHC class Iallele can be used to determine what alleles are expressed.Alternatively, PCR amplification of nucleic acids encoding MHC class Ialleles can be used.

By “syngeneic donor cell” or “isogeneic donor cell,” is meant (i) adonor cell that is genetically identical, or matched at the HLA region(i.e., has at least four, and preferably all 6, of the standard markersin common with), to a cell of the recipient or (ii) a donor cell that isre-administered to the same patient from which it was obtained.

A “TNF-alpha inducing agent,” is desirably a compound that results inthe expression of endogenous TNF-alpha, enhances secretion of TNF-alpha,or enhances bioavailability or stability of TNF-alpha. However,TNF-alpha agonists, agents that stimulate TNF-alpha signaling or enhancepost-receptor TNF-alpha action, or agents that act on pathways thatcause accelerated cell death of autoimmune cells, are also included inthis definition. Stimulation of TNF-alpha induction (e.g., byadministration of CFA) is desirably carried out prior to, after, orduring administration (via implantation or injection) of cells in vivo.

By “lymphopenia” is meant a decrease in the total number of lymphocytesin a blood sample from a mammal. In some embodiments, this decrease isdue to death of lymphocytes, such as T-cells, B-cells, and/ormacrophages. In certain embodiments, this decrease is due toredistribution of lymphocytes.

By “nonspecific,” in reference to lymphopenia, is meant a reduction inthe total number of lymphocytes in an individual, and is not limited toa subset of lymphocytes.

By “selectively killing blood cells” is meant directly or indirectlyreducing the number or relative percentage of a subpopulation of bloodcells (e.g., autoreactive lymphoid cells such as T- or B cells or thedefective antigen presenting cells) such as a subpopulation ofunstimulated cells or stimulated cells. In desirable embodiments, thesubpopulation is a subset of T-cells, B-cells, or macrophages.Desirably, the killed memory T-cells are autoimmune T-cells, i.e.,T-cells that are activated by presented self epitopes. In desirableembodiments, the killed naïve T-cells are cells that would otherwisebecome autoimmune T-cells. Desirably, the number of autoimmune T-cellsor cells that would otherwise become autoimmune T-cells decreases by atleast 25, 50, 100, 200, or 500% more the number of healthynon-autoimmune T-cells decreases. In some embodiments, the number ofautoimmune T-cells or cells that would otherwise become autoimmuneT-cells decreases by at least 25, 50, 75, 80, 90, 95, or 100%, asmeasured using standard methods. The T-cells can be killed due to anypathway, such as apoptosis, necrosis, and/or activation induced celldeath. Apoptosis can be assayed by detecting caspase-dependent cellshrinkage, condensation of nuclei, or intranuclear degradation of DNA.Necrosis can be recognized by caspase-independent cell swelling,cellular degradation, or release of cytoplasmic material. Necrosisresults in late mitochondrial damage but not cytochrome C release. Insome embodiments, memory T-cell are killed by apoptosis, and naïveT-cells are killed by necrosis. For the treatment of lupus, B-cells aredesirably killed or, alternatively, they are allowed to developmentallymature.

By “stimulated blood cell” is meant a blood cell (e.g., a memory T-cell,a B-cell, or a macrophage) that has been exposed to an antigen.

By “unstimulated blood cell” is meant a blood cell (e.g., a naïveT-cell, a B-cell, or a macrophage) that has not been exposed to anantigen.

By “pathologic T cell” is meant a T cell that is involved, or has thepotential to be involved, in an autoimmune response or disorder.

Stimulated cells tend to be in later stages of maturation thanunstimulated cells, in active progression through the cell-cycle, and/orinvolved in infiltrating a diseased or damaged organ or tissue.Unstimulated cells tend to progress through the cell-cycle more slowlyor not at all. Memory T-cells tend to express a higher density of IL-2receptor (e.g., 10-20% higher density) than naïve T-cells. Naïve T-cellstend to express a higher density (e.g., a 5-20% higher density) ofCD45RB^(high), CD95, and/or CD62L than memory T-cells.

By “purified” is meant separated from other components that naturallyaccompany it. Typically, a factor (e.g., a protein, small molecule, orcell) is substantially pure when it is at least 50%, by weight, freefrom proteins, antibodies, and naturally-occurring organic moleculeswith which it is naturally associated. Desirably, the factor is at least75%, more desirably, at least 90%, and most desirably, at least 99%, byweight, pure. A substantially pure factor may be obtained by chemicalsynthesis, separation of the factor from natural sources, or productionof the factor in a recombinant host cell that does not naturally producethe factor. Proteins, vesicles, chromosomes, nuclei, other organelles,and cells may be purified by one skilled in the art using standardtechniques such as those described by Ausubel et al. (Current Protocolsin Molecular Biology, John Wiley & Sons, New York, 2000). The factor isdesirably at least 2, 5, or 10 times as pure as the starting material,as measured using polyacrylamide gel electrophoresis, columnchromatography, optical density, HPLC analysis, or western analysis(Ausubel et al., supra). Preferred methods of purification includeimmunoprecipitation, column chromatography such as immunoaffinitychromatography, magnetic bead immunoaffinity purification, and panningwith a plate-bound antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains drawings executed in color (FIG. 1).Copies of this patent or patent application with color drawings will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1 is a table that demonstrates the presence of Y-chromosomepositive cells in adult female NOD mouse pancreatic islets with diseasereversal. Column 1 and column 2 represent two different female NOD micewith long term correction of their disease defined as the reversal ofhyperglycemia followed by regeneration of the insulin-secreting cells inthe pancreas. All histology sections from each animal were stained byimmunohistochemistry and were from consecutively cut specimens. Thefirst row shows the stain of the pancreas with insulin antibodies,demonstrating the reappearance of beautiful islets free of disease. Thesecond row demonstrates that Y-chromosome staining of the same isletproduces punctuate dark pink staining of the Y chromosome only withinthe islet tissue and associated duct tissue, but not within theassociated exocrine portions of the pancreas. This result is definitiveevidence that the origin of the reappearing islets is the male donorsplenocytes. Row 3 is a close-up picture of the associated duct thatalso shows that the Y cells were not only present in the islet but alsopopulate the duct tissue. The duct was repopulated by the donor cellsand is less uniform than the islet repopulation. In general, almost allthe islet tissue in the pancreases of these mice was of donor origin, asdemonstrated by the uniform Y chromosome presence in all cells. Theducts in the pancreases of these animals may be of completely femaleorigin or of a mixed origin composed of both female and male cells. Row4 demonstrates that a developmental marker of early islet regenerationis PDX-1. The staining of the pancreatic sections reveals bright PDX-1staining, exclusively in the regenerating islet but not in theassociated exocrine portions of the pancreas.

FIGS. 2A-2C are graphs demonstrating the percentage of CD8CD45RB^(high), CD8 CD62L, and CD8 CD95 cells in mice treated with orwithout CFA and with or without donor splenocytes.

FIGS. 3A and 3B are graphs of the percent change in the mean antigendensity in mice treated with or without CFA and with or without donorsplenocytes.

FIG. 4 is a table demonstrating the ability of multiple doses ofTNF-alpha to inhibit insulitis in 20 to 35 week old, diabetic mice.

FIG. 5 is a list of exemplary death receptors.

FIG. 6 is list of exemplary inhibitors of cell death that can be used toprevent death of transplanted donor cells or of endogenous healthy cells(Oncogene Research Products, 2002/2003 catalogue, San Diego, Calif.). Anexemplary inhibitor of necrosis is geldanomycin, and an exemplaryinhibitor of apoptosis is zVAD-fmk.

FIG. 7 is a list of standard kits that can be used to measure the levelof cell death (Oncogene Research Products, 2002/2003 catalogue, SanDiego, Calif.).

FIG. 8 is list of exemplary compounds that induce cell death (OncogeneResearch Products, 2002/2003 catalogue, San Diego, Calif.).

FIG. 9 is a table that summarizes the analysis of splenocytes or PBLsfrom mice injected biweekly for 40 days with 10⁷ donor cells. Fewercells may also be used in each injection in the present methods,especially if the cells are administered more often and/or for longer.Column “Whole GFP+%” lists the percentage of splenocytes or PBL cellsthat are fluorescent, indicating they are of donor origin. Column “H-2kb+GFP+” lists the percentage of analyzed cells that are fluorescent andthat express the same class 1 locus as the donor cells. Column “CD3+ +GFP+” lists the percentage of analyzed cells that are fluorescent andexpress CD3, which is only expressed on T-cells. Column “B220+ GFP+”lists the percentage of analyzed cells that are fluorescent and expressB220, which is only expressed on B-cells. Column “Mac-1+GFP+” lists thepercentage of analyzed cells that are fluorescent and express Mac-1,which is only expressed on macrophages.

FIG. 10 is a table that summarizes characteristics of mice treated withvarious methods of the present invention. “TNF-α low dose” refers to a 2μg dose of TNFα. “Sp” denotes splenocytes, and “Bm” denotes bone marrowdonor cells. “(−)” indicates no significant difference compared tountreated control mice, “(+),” “(++),” and “(+++)” denote increasingdifferences (e.g., increased activity or increased number of cells)compared to untreated control mice.

FIG. 11 is a table summarizing conditions that promote regeneration.

DETAILED DESCRIPTION

We have succeeded in regenerating a functioning organ (the islet cellsof a pancreas) in an animal with a damaged pancreas, and have shown thatalmost all of the visible portions of the organ, as well as a largeportion of the bile ducts, are of donor origin.

The present methods for increasing the number of functional cells of apredetermined cell type in a patient have a number of advantages. Forexample, the present methods are robust and durable. The methods can beused to replace most or all of an endogenous organ using donor cells ofthe same cell type or of a different cell type as the organ. The methodscan also be performed without immunosuppression for nonspecificinactivation or death of autoimmune cells.

These following examples are provided for the purpose of illustratingthe invention and should not be construed as limiting.

Materials and Methods

Diagnosis and Treatment of Mice

Female NOD mice were obtained from either Taconic Farms (Germantown,N.Y.) or Jackson Laboratory (Bar Harbor, Me.). C57BL/6J mice wereobtained from The Jackson Laboratory. All mice were maintained underpathogen free conditions.

NOD mice were screened for the onset of diabetes by monitoring theirbody weight and blood sugar level. The criteria for diagnosis ofdiabetes included two consecutive blood sugar concentrations exceeding400 mg/dl. Mice with blood sugar levels greater than 400 mg/dl weregiven daily injections of 1.0 to 1.5 units of Neutral Protamine Hagedorn(NPH) human insulin for each 100 grams of body weight to preventimmediate death due to hyperglycemia. The use of such severely diabeticmice relatively late after disease onset ensures that the endogenouspancreas islets were completely obliterated prior to transplantation ofthe donor islet cells.

Splenocyte donors included normal C57 mice, C57 β₂M^(−/−) mice whosecells have a decreased ability to express MHC class I and peptide ontheir surface due to the ablation of the chaperone protein β₂microglobulin but can express MHC class I and self peptide when exposedto normal mouse serum, C57 β₂M^(−/−) TAP1^(−/−) mice which have adecreased ability to re-express MHC class I and self peptide, and MHCclass II^(−/−) mice in which the I-A gene is disrupted and the E locusfor MHC class II is not expressed because of endogenous defects in theC57 strain. Splenocytes, generally at a dose of 9×10⁶ cells, wereinjected into some NOD recipients through the tail vein twice a week.TNF-alpha, which is commercially available from a variety of vendorsincluding Genentech (South San Francisco, Calif.), Hoffman-LaRoche(Basel, Switzerland), Boehringer Ingelheim (Germany), Asahi ChemicalIndustry (Japan), and Sigma-Aldrich (St. Louis, Mo.), was administeredintraperitoneally (1-20 μg per dose, 2 to 3 times per week) to eliminateautoreactive cells and promote regeneration in the host byrecapitulating the embryonic program of blood vessel endothelium, i.e.induction of NF-B and/or VEGF. CFA (Difco Laboratory, Detroit, Mich.)was mixed with an equal volume of physiological saline, andapproximately 1-50 μl were injected into each hind-footpad at the timeof the islet transplantation or after the first splenocyte injection.

Islet Transplantation

Islets were isolated from donor C57 mice or 6 to 8 week-old pre-diabeticfemale NOD mice and served as a glucose clamp and the source of MHCclass I and self-peptide. Gradient centrifugation followed by manualselection of islets was performed to ensure that both preparations werehighly enriched for islets and to accurately determine the number oftransplanted islets. Approximately 500 to 600 specially selected isletswere grafted beneath the left kidney capsule of each diabetic NODrecipient. For islet encapsulation, 900-1100 islets were enclosed in0.2-0.5 cm diameter alginate spheres that were surgically inserted intothe peritoneal cavity of diabetic NOD mice. Transplantation wasconsidered successful if the non-fasting blood glucose concentrationreturned to normal within 24 hours after surgery. The glucoseconcentration of the recipient's blood was monitored three times a weekafter transplantation with a Glucometer Elite instrument (Bayer Corp.,Pittsburgh, Pa.). Allografts were considered to have been rejected ifthe blood glucose concentration increased to more than 250 mg/dl at twomonitoring time points. The recipients that rejected allografts weretagged for histological examination and flow-cytometric studies.

To determine the effect of the endogenous pancreatic islets in thecontrol of blood sugar concentration, the subrenal islet transplantswere surgically removed and analyzed. Similarly, islets that had beenencapsulated in alginate spheres were removed, as necessary, from theperitoneal cavity after direct localization using a dissectingmicroscope. Histological analyses of the pancreata and allograft wereperformed by (i) staining with hematoxylin and eosin for evaluation oflymphoid infiltrates and (ii) staining with aldehyde-fuchsin for insulinislet content. The entire pancreas from splenic to duodenal stomachattachments was removed, fixed, and subjected to serial sectioning,usually at 10 μm per section.

Example 1 CFA Treatment and Islet Transplantation in NOD Mice

Hosts for the transplantation experiments were severely diabetic femaleNOD mice, usually greater than 20 weeks of age, which exhibited bloodglucose concentrations of greater than 400 mg/dl for at least seven daysand had been treated by daily administration of insulin for that lengthof time. Islet transplants were placed unilaterally in the kidneycapsule to facilitate non-lethal removal and histological examination.Islets from 6-8 week-old pre-diabetic donor females or from normal C57donor mice were rapidly rejected by diabetic NOD hosts in all cases,usually by day 9. Although C57 donor islets with transient ablation ofclass I survive indefinitely in non-immunosuppressed diabetic andnon-autoimmune diabetic hosts, the survival time for β₂M^(−/−) C57islets in diabetic NOD females is only about two to three times that ofnormal C57 islets. This observation confirms that the ablation of thedonor cells is related to the current disease, and not related toallograft rejection. CFA treatment prolonged survival of syngeneic isletgrafts in diabetic NOD hosts, but had a minimal effect on the survivalof C57BL/6 islets, which were uniformally rejected about 11 days aftertransplantation. However, the combination of β₂M C57 islet transplantswith CFA treatment resulted in sustained normoglycemia for more than 120days in 5 out of 14 diabetic hosts. Although the duration ofhyperglycemia before initiation of the therapy varied between 7 and 20days in these cohorts, the length of this interval was not statisticallyrelated to the duration of sustained normoglycemia after treatment. Theanimals in this study with sustained normoglycemia also had progressiveweight gain similar to that in NOD female host cohorts that never becamediabetic. The normalization of blood sugar concentration is a measure oftreatment success.

After the recurrence of hyperglycemia in NOD mice that had been treatedwith CFA and syngeneic NOD transplants that do not have MHC classI-presented self-peptide, the kidney containing the allograft wasexamined histologically. Macrophage and T-cell specific infiltrates wereapparent under the kidney capsule at the site of transplantation, acharacteristic of recurring autoimmune disease. Moreover, no intactislets were detected in the pancreas. Although an occasional host hadislet remnants in the pancreas, these were largely obscured by densepockets of infiltrating lymphocytes. This recurrence of hyperglycemiaafter administration of NOD transplants may be due to the inability ofthe MHC class I in the NOD transplants to present peptide because of anerror in this pathway in NOD mice. This lack of peptide presented by MHCclass I cells may prevent the transplanted cells from inducingtolerance. Alternatively, even if NOD islets present MHC class I andpeptide, it is possible that a single exposure of a parenchymal cell isinsufficient. Thus, the host NOD mice generate autoimmune T-cells thatdestroy the transplanted cells.

Similar histological characteristics, including infiltrating lymphocytesat both the transplant site and in the pancreas, were apparent indiabetic NOD mice that received CFA in combination with semi-allograftsfrom C57 donors. Unexpectedly, for all NOD mice with long-termnormoglycemia, after removing the β₂M^(−/−) C57 islets, and after CFAtreatment, no surviving allografts were detected under the kidneycapsule when the animals were examined more than 129 days aftertransplantation using this protocol. In contrast, the pancreas in eachof these five recipients exhibited well-formed islets that appearedcompletely granulated when stained with aldehyde-fuchsin. Our resultsshow that the islets were free of lymphocytes, with lymphocytes onlypresent around the circumference of the islets. This pattern oflymphocyte accumulation, with lymphocytes surrounding, but not invadingthe islets has been associated with non-progressing or interrupted betacell autoimmunity. The return to normoglycemia in the absence ofdetectable transplanted islet tissues, together with the presence ofislets in the pancreas largely devoid of infiltrating lymphocytesindicates not only that autoimmunity has been interrupted, but also thatthe function of the endogenous beta cells had been restored. Thisdisruption of autoimmunity is thought to be due, at least in part, tothe induction of TNF-alpha by the administered CFA, and the destructionof autoimmune T-cells by the resulting TNF-alpha. Additionally, theexpression of MHC class I with an allele that matches the host andpeptide by the donor cells induces tolerance by T-cell re-selectionperhaps both by necrosis and apoptosis that also prevents destruction ofthe transplanted cells and regenerating organ.

The relative contribution of endogenous cells and donor cells torestoring endogenous beta cell activity was measured to determinewhether the increase in beta cell activity was a result of endogenousregeneration from host precursor cells or, as demonstrated below, theconversion of donor lymphoid cells or donor islets into hosts islets. Asdescribed below, although rescue may play a role in the reversal ofearly diabetes and late diabetes, the administered donor cells werecells that could become host pancreatic beta cells. Restoration of nearnormal pancreatic islet histology was observed only in diabetic NOD micethat received both the β₂M^(−/−) allograft and the CFA treatment.Pancreatic islets were not detected in any diabetic NOD mouse treatedwith CFA and syngeneic NOD islets. The persistence of normoglycemia inrecipients of syngeneic NOD islets was apparently solely due totransplanted islets which always exhibited invasive insulinitis. Here,treatment with CFA, together with syngeneic NOD islets may have sloweddisease recurrence, but persistent autoimmunity remained. We alsoassessed the relative contribution of restored endogenous pancreaticislets and transplanted islets to the maintenance of normoglycemia inNOD mice treated with CFA and allografts from β₂M^(−/−) C57 donors. Therelative contribution of the endogenous pancreatic islets was determinedby removing the kidney containing the islet transplants after 120 daysof normoglycemia from a group of five animals, as well as from a controlgroup that had not received the allograft. All five mice which hadreceived the allograft remained normoglycemic after nephrectomy untilthey were killed 3-60 days later. Histological analysis of kidneys thathad not received the graft revealed a complete loss of identifiableislet structures. In contrast, the pancreata for all five allograftrecipients contained well-formed islets, either without lymphoidinfiltrates or with circumferentially distributed lymphocytes only.Normoglycemia after nephrectomy was thus maintained solely by endogenouspancreatic islet that we now know had reappeared in the host by bothrescue as well as regeneration from endogenous or exogenous sources.

These results were affirmed by analysis of nephrectomies performedduring the post-transplant period of normoglycemia on two mice which hadreceived CFA plus syngeneic NOD islets. In this case, the treatmentresulted in a rapid return to hyperglycemia, demonstrating that thecontrol of blood sugar in this treatment group was mediated solely bythe transplanted islet tissue. Diabetic NOD mice were also transplantedwith islets from C57 mice in which the genes from both the β₂M and TAP1genes had been deleted. Together with TAP2, TAP1 mediates transport ofendogenous peptides from the cytosol into the lumen of the endoplasmicreticulum for the assembly with MHC class I molecules. Our data usingC57 mice in which the genes for both β₂M and TAP1 had been deletedshowed that these mice are more defective in the presentation of MHCclass I self-antigens than those only having a mutation in the β₂M gene.Transplantation of these double-knockout C57 islets lacking both β₂M andTAP1, combined with the injection of CFA results in return ofhyperglycemia within 14 days in some of the animals. Two out of six NODmice treated with the double knock out Class I cells had normoglycemiaafter 40 days of treatment. Physiological examination of the pancreasrevealed a pattern typically seen in untreated diabetic NOD mice. Thedecrease in efficiency of this protocol supports the important role ofcorrectly assembled and administered MHC class I and peptide complexes.

Based on these results, a transient interruption of peptide presentationof donor MHC molecules or a transient deficient MHC class I is importantfor the abrogation of autoimmunity. This transient ablation or decreasedMHC class I or permanent presentation of donor MHC class I also appearsto be a feature that allows these cells to transform into other celltypes. In contrast, the sustained interruption of this process preventsthe re-establishment of tolerance and the restoration of endogenouspancreatic islet integrity. The repeat administration of a normal MHCclass I and self peptide or a short lived cell showed similar efficacy.

Given that the restoration of normoglycemia in diabetic NOD hoststreated with CFA and β₂M^(−/−) C57 islets cannot depend on thecontinuing secretion of insulin by the islet graft, we now investigatedwhether C57 donor cell types, other than islets, might serve atherapeutic role. We initially performed these experiments expecting theC57 donor cell to provide MHC class I and self-peptide. As shown by thefollowing results, donor lymphoid cells actually transform into insulinsecreting beta cells in the host or fuse with damaged cells of the hostor adjacent cells of the host. In these experiments, nine diabetic NODmice were treated with a single bilateral injection of CFA, followed bya 40-day regimen of biweekly intravenous injections of C57 splenocytes.These lymphoid cells express both MHC class I and MHC class II proteinsand survive only transiently in NOD hosts because of presumed graftrejection. However, contrary to the prior assumption that this would beonly a transient treatment, these donor cells survive long-term withouta need for immunosuppression in the host. Repeat injections ofsplenocytes ensured that the host was continuously exposed to antigenpresentation complexes on the surface of these cells. Recipients weremonitored for hyperglycemia every 3-4 days and insulin was administereddaily unless normoglycemia returned. A control group of four diabeticNOD mice received daily insulin injections only. All four control groupmice died on or before day 25 of the experimental period as a result ofpoor control of blood sugar and consequent ketoacidosis. In contrast,some of the mice injected with CFA and C57 splenocytes were alive after40 days, and three of these animals had become normoglycemic and insulinindependent.

While the pancreata of control mice exhibited pronounced lymphocyticinfiltrates that obscured any remaining islet structures, the pancreataof the four NOD mice that were treated with CFA and C57 splenocytes andremained alive but hyperglycemic and insulin dependent revealed a markeddecrease in the number of lymphoid infiltrates located circumferentiallyor adjacent to the infrequent islet structures. In the three NOD micetreated with C57 splenocytes and CFA that remained normoglycemic afterthe discontinuation of insulin injections, the pancreata exhibitedabundant islets that were free of invasive insulinitis or isletsassociated only with circumferential lymphocytes. The treatment of CFAcombined with re-exposure to C57 lymphocytes resulted in completereversal of diabetes in approximately 30% of NOD recipients and partialrestoration of beta cell function in approximately an additional 40% ofhosts.

Subsequent experiments were conducted to determine whether theefficiency of the system could be improved by regulating glycemiccontrol in the host. The reversal of diabetes in NOD mice by CFA andrepeat exposure to C57 splenocytes indicated that the restoration ofendogenous islet function is achievable without islet transplantationand despite the poor glycemic control attained by insulin injection. Todetermine whether the restoration of endogenous beta cell function couldbe achieved more consistently with better control of blood glucose,insulin injections were replaced with the intraperitoneal implantationof alginate encapsulated C57 mouse islets. Alginate encapsulationprevents direct contact between the donor tissue and the host T-cellsand such grafts have been shown to provide near normoglycemic controlfor 40 days in approximately 78% of autoimmune NOD recipients. Almostall diabetic NOD mice that received alginate encapsulated C57 isletsexhibited improved glucose regulation or normoglycemia. The alginatespheres were removed 40-50 days after implantation, and the bloodglucose concentration was monitored. The seven mice treated only withalginate encapsulated islets, the six mice that received a singlebilateral injections of CFA, and the three mice treated with biweeklyinjections of C57 splenocytes, all exhibited a rapid return tohyperglycemia and early death after removal of the implants. Pancreataof NOD mice that received only alginate encapsulated islets revealed nosign of intact islets or of lymphoid infiltrates. The pancreata of NODhosts treated with CFA and alginate encapsulated islets exhibited markedinvasive insulinitis and obscured islet structures. In contrast, sevenof the nine diabetic NOD hosts that received CFA and C57 splenocytesremained normoglycemic for more than 40 days after removal of thealginate encapsulated islets. The pancreata of these animals containedlarge islets with circumferentially distributed lymphocytes. The isletmass after at least 80 days of disease reversal was estimated to beapproximately 50% of the original value. The pancreata from controlBALB/C mice contained approximately 25-35 islets, and the pancreata fromsuccessfully treated NOD mice contained approximately 12-20 islets, asdetermined by serial histological sectioning.

In addition, the maintenance of normoglycemia due to the treatmentincreased the percentage of diabetic mice permanently cured ofhyperglycemia. We next identified features of this treatment regimenthat contributed to the production of a positive outcome. As is notedabove, CFA was used to induce the endogenous production of TNF-alpha, aswell as other cytokines believed beneficial for removal of autoimmunityand to promote regeneration. The role of TNF-alpha in the treatment ofdiabetes was therefore investigated by the intravenous administration ofrat IgG1 monoclonal antibodies to the cytokine TNF-alpha at a dose ofapproximately 1.5 mg/day for the first 10 days in diabetic NOD hoststreated with C57 splenocytes, CFA, and alginate encapsulated islets. Allfive NOD mice so treated exhibited a rapid return to hyperglycemia uponremoval of the alginate encapsulated islets 50-70 days aftertransplantation consistent with the role of TNF-alpha in the beneficialeffect of CFA. In a related experiment, an anti-TNF-alpha antibody(clone MP6-X73) was administered at a dose of 1.5 mg/day for 10 daysafter administration of CFA, C57 splenocytes, and alginate encapsulatedislets into diabetic NOD mice (n=5). After removal of thealginate-encapsulated islets at day 40, hyperglycemia returnedimmediately in all five mice. In mice treated similarly except for theadministration of the anti-TNF-alpha antibody, normoglycemia wasmaintained in seven out of nine NOD mice after removal of the alginatebeads. The specificity of the effect induced by the anti-TNF-alphamonoclonal antibody was confirmed by the failure of the control rat IgG1monoclonal antibody reactive with human T-cell receptor beta 1 chain toproduce a therapeutic effect.

To demonstrate an increase in TNF-alpha levels due to administration ofCFA, levels of TNF-alpha were measured in NOD mice after a singleinjection of CFA with or without donor lymphocytes. As Table 1, slightlyelevated levels of TNF-alpha were transiently detectable in NOD mice for2-5 days after a single injection of CFA with or without splenocytes.NOD mice at days 2-8 after a single dose of CFA also have decreasedplatelet levels from 30,000-60,000/mm³. This data indirectly supportsCFA's induction of TNF-α in NOD mice.

TABLE 1 Treatment of NOD mice with CFA results in elevated plasmaTNF-alpha. Donor TNF (g/mL) Group Cells CFA Day 0 Day 2 Day 5 Day 21 Day40 1 — − .40 .35 .41 .42 .38 2 — + .51 20.1 18.1 .49 .30 3 C57BL/ + .3521.7 17.5 .50 .57 6 TNF-concentrations were measured by solid phaseELISA using a sandwich technique with two different monoclonalantibodies to mTNF-alpha, one of which was conjugated to horse radishperoxidase (Sigma, St. Louis, MO). The limit of detection was 0.2units/ml.

The data presented above show that the injection of CFA, the endogenousinduction of TNF-alpha, or the administration of TNF-alpha directlyresults in the permanent elimination of TNF-alpha sensitive cells, themajority of which have previously seen islet cell antigens. In additionto the beneficial apoptotic death or effect on the lymphoid system, wealso demonstrated that TNF-alpha binds directly to TNF Receptor 2 on thevascular endothelium, the complementary matrix for differentiation, andthe islet precursor cells or islet beta cells themselves, therebypossibly also promoting regeneration in the pancreas. We further showedthat the introduction of MHC class I peptide complex expression, eitheron the surface of normal islet cells or normal lymphocytes, results in apartial, but stable, reselection of T-cell population from the NOD hostsleading to an increase in the abundance of long-term memory cells. MHCclass I and self-peptide reintroduction is important for the reselectionof cells that probably have less stimulation with islet cell antigen,but the equivalent potential for autoreactivity (see, for example, U.S.patent application Ser. No. 09/521,064, filed Mar. 8, 2000, and09/768,769, filed Jan. 23, 3001, and PCT publication WO00/53209,published Sep. 14, 2000, which are each incorporated by reference).Furthermore, additional studies presented herein show that these cellsactually persist long-term in the host. We demonstrate that without anyimmunosuppression to the host, the semi-allogeneic MHC class Isplenocyte origin cells or splenocyte residing cells, as well assemi-allogeneic cells from spleens differentiated into islets, persistlong-term in the host without the need for immunosuppression.Furthermore, the reversal of any poorly controlled metabolic conditionappears to promote the regenerative process and the reversal ofestablished disease.

Example 2 Optimizing a Curative Therapy in the Diabetic NOD Mouse

In order to examine the differential effects of CFA, BCG, TNF-α, andsplenocyte administration, late stage NOD mice (>15 weeks of age) wererandomly assigned to one of four treatment groups, i.e., one injectionof CFA, one injection of BCG (4 mg/kg), one injection of 10 ug TNF-α, orone injection of F1 splenocytes (1×10⁶ cells, IV) obtained from normaldonors. The treated NOD mice were serially sacrificed on day 2, day 7,and day 14 (Table 2). An examination of pancreatic histology evaluatedthe effects of the interventions on invasive insulitis (Table 3).

Table 2 shows that on day 2 both a single injection of CFA and a singleinjection of low dose TNF-α (10 g) had eliminated completely allsubpopulations of cells with in vitro TNF-α sensitivity. At day 7 andday 14, this population of TNF-α sensitive cells was again evident.Simultaneous pancreatic histology of these cohorts confirmed a dramaticreduction in insulitis, as well as a lingering effect lasting beyond day14 with respect to insulitis (Table 3).

TABLE 2 Percentage of remaining TNF -apoptotic-sensitive NOD splenocytesafter various in vivo treatments Day 0 (%) Day 2 (%) Day 7 (%) Day 14(%) NOD mice 0 ng 20 ng 0 ng 20 ng 0 ng 20 ng 0 ng 20 ng CFA 26.6 2429.2 34.2 26.6 35.1 10 g m-TNF 27.9 28.2 28.9 36 BCG 7.4 22.3 15.3 26.737.8 28.9 F1 splenocytes 4.3 27.5 3.8 23.1 0.8 33.7 untreated 22.1 32.627.1 41.8 30.7 41.7 29.4 38.6 Day 0 Day 2 Day 7 Day 14 C57BL/6 0 ng 20ng 0 ng 20 ng 0 ng 20 ng 0 ng 20 ng untreated 14.1 14.7 12.3 12.8 18.816.2 17.5 17.2 NOD mice in this study were in a late pre-diabetic stageof disease at 18 weeks of age with at least one blood sugar greater than200 mg/dL. Late apoptotic cells by flow cytometric studies of NOD micetreated with various immunomodulatory interventions were quantified onsplenocytes after animal sacrifice in the days indicated after treatmentinitiation. For these flow cytometric studies, late apoptosis representsAnnexin V+ PI+ and Annexin V+ PI− cells after 24 hours in vitro exposureto TNF- (20 ng/mL)

TABLE 3 Percentage of NOD islets with remaining invasive insulitis aftervarious in vivo treatments Day 0 (%) Day 2 (%) Day 7 (%) Day 14 (%) CFA4 22 10 mg m-TNF 7 33 BCG 9 67 F1 splenocytes 26 31 untreated 100 0 100100 The mice in Table 3correspond to the same mice shown in Table 2

The responses to F1 splenocytes and BCG were markedly different thanthat seen with CFA and TNF-α, and somewhat different from each other.The therapeutic effect of F1 splenocytes was an elimination of the NODlymphoid cells, which we believe represent pathogenic naïve cells,perhaps through a direct or indirect mechanism (Table 2). TNF-αsensitivity remained and the F1 splenocyte therapeutic impact lastedbeyond day 14. The histology revealed a less dramatic impact on thenumbers of NOD islets with remaining invasive insulitis. In addition, F1splenocytes eliminated “cords” of invasive insulitis per islet insteadof the more homogenous central elimination of insulitis characteristicof either TNF-α or CFA (not shown).

Unexpectedly, BCG, a known inducer of TNF-α had a greater impact on theelimination of tissue culture (naïve) sensitive, pathogenic cells thanit had on the elimination of pathogenic memory cells. The therapeuticeffect of a single, low dose injection of BCG waned rapidly over thetime course of 14 days (Table 2). The histologic analysis of the NODpancreases confirmed partial elimination of insulitis (Table 3). Similarto the end result when MHC class I and self-peptide were reintroduced,the tissue culture-sensitive subpopulation of cells was eliminated.

These data are consistent with data in our earlier publication relatingto the use of two limbs of interventions to “reverse” disease in NODmice (i.e., TNF-α and F1 splenocytes). Our data show that theseinterventions can produce a distinct, measurable impact on specificlymphoid cell populations in the spleen. Each of the two limbs appearsto target a different subpopulation of pathogenic cells with heightenedapoptosis sensitivity. Importantly, the changes in T cell response toculture and TNF-α in vitro parallel the pancreatic histology of reducedinvasive insulitis.

Although initially thought to be due to its reported induction ofendogenous TNF-alpha release, the action of BCG in NOD mice appears tobe predominately due to an indirect impact on naïve cell selection bythe direct killing of monocytes/macrophages that have defective MHCclass I presentation and, to a lesser extent, by reduction of themeasurable burden of memory cells with TNF-alpha sensitivity. BCG isknown to infect macrophages/monocytes and, if the BCG is avirulent,induces lysis of the macrophage/monocyte, thus reducing hamperingcontinued production of tuberculin particles. Macrophage/monocyte lysisreleases TNF locally, with infected cells usually containing abundantintracytoplasmic concentrations of this cytokine. In the NOD mouse, theavirulent strain of BCG causes lysis of certain subpopulations ofmonocytes/machrophages in an accelerated fashion. These cells appear tobe developmentally “immature”, with lower levels of MHC class I-selfpeptide expressed on the surface. This suggests that BCG sub-strainswith the lowest virulence, derived from the in vitro selection of thosethat most rapidly lyse human or murine autoimmune monocytes, would bethe best BCG strains to treat autoimmune disorders. Furthermore, thesedata suggest three strategies for identifying and eliminating naïve Tcells: direct death receptor stimulation of the T cells through asusceptible receptor on the cells; introduction of corrected antigenpresenting cells or complexes; with self peptide and or self peptide/MHCclass I complexes, or the introduction of agents or adjuvants like BCGthat cause the direct death of the monocytes/macrophages with the mostsevere defects in antigen presentation/processing, thereby eliminatingthe defective educator cells. In all three strategies, endogenousmonocytes/macrophages with sufficient antigen presentation to bias the Tcell repertoire back towards normal reselection predominate.

Example 3 Functional Impact of Donor Cell Radiation on Restoration ofNormoglycemia in Severely Diabetic NOD Mice

Severely hyperglycemic NOD mice were originally treated with CFA toinduce TNF-alpha and simultaneously exposed to functional complexes ofMHC class I molecules and allogeneic peptides presented on either viablesplenocytes or on islets. Normoglycemia for a 40-day treatment period, acritical parameter of this approach, was induced either by theimplantation of temporary alginate encapsulated islets or subrenallyplaced syngeneic islet transplants. Both methods of glucose control canbe surgically removed to test for restored endogenous pancreaticfunction. All original experiments were designed to treat establishedNOD female mice with severe hyperglycemia and utilized a 40-day timeperiod of tight, artificial glucose control. Utilization of thistemporary glucose control permitted sufficient endogenous pancreaticre-growth of the endogenous pancreas to sustain normoglycemia in up to78% of formerly hyperglycemic NOD cohorts and to restore endogenouspancreatic insulin secretion.

To dissect the mechanism to NOD disease reversal, irradiated donor cellsand live cells expressing MHC class I and self-peptide were studied.These experiments tested the role of long-term donor cell survival andthe role of the donor cell function (e.g., antigen processing) in thepermanent reversal of tolerance for self-antigens. NOD hosts used inthese experiments were severely diabetic NOD mice typically greater than20 weeks of age that exhibited blood glucose concentrations of greaterthan 400 mg/dl for at least seven days. All diabetic NOD hosts at thislate stage of disease were dead within two weeks because of the severityof the disease. Alginate encapsulated islets were implanted into theperitoneal cavity for 40 days, and the hosts were randomized to receiveeither CFA alone or CFA in combination with intravenous biweeklyinjections of irradiated C57BL/6 splenocytes. At this point, the C57BL/6splenocytes are only semi-allogeneic to the NOD host; the splenocytesare H2K^(b)D^(b), and the NOD mouse is H2K^(d)D^(b).

As we previously demonstrated, new cohorts of NOD mice immunized withlive C57BL/6 splenocytes demonstrated restored normoglycemia. Seven ofthe nine newly treated NOD cohorts remained normoglycemic after thesurgical removal of the alginate encapsulated C57 mouse islets. Thereversal of autoimmunity was permanent, and the seven NOD hosts remainedeuglycemic for observation periods beyond 80 days at which time theywere sacrificed. Eight additional NOD mice were treated with the sameregimen, but with irradiated donor C57BL/6 splenocytes. In all eightcases, hyperglycemia returned within two to seven days after the removalof the alginate encapsulated islet that served as a glucose clamp duringthe 40-day treatment period. Pancreata of NOD mice that received liveC57 splenocytes revealed abundant islets in seven of the nine mice. Nosigns of invasive islet lymphoid infiltrates, which are a sign of activeautoimmunity, were present. When an islet was present, fewer than 10% ofislet circumferential lymphoid cells encircled the pancreatic islet.Surprisingly, the pancreata of NOD mice that remained hyperglycemic andreceived irradiated donor splenocytes consistently revealed islets inthe pancreas, but with decreased overall abundance as assessed by serialpancreatic sections. The islets in these pancreata were accompanied withsizable circumferential islet infiltrates but lacked lymphocytes withinthe islet structure itself, a histological pattern referred to asinvasive insulinitis. For instance, pancreata from NOD mice treated withlive splenocytes and having restored normoglycemia containedapproximately 6-8 islets with each serial histological section, whilepancreata from NOD mice treated with irradiated splenocytes and havingpersistent hyperglycemia contained 4-6 islets per serial histologicalsection. Both irradiated and live splenocytes appeared to rid thediabetic host of invasive and destructive insulinitis. However,increased islet abundance and restored insulin secretion were onlyobserved in NOD cohorts treated with live splenocytes.

In evaluating the significance of apparent histological elimination ofdestructive insulinitis without sufficient insulin secretion after theintroduction of irradiated donor cells expressing MHC class I andself-peptide, additional diabetic NOD mice were treated. For theseexperiments, we used a treatment period of 40 days of donor MHC class Iand self-peptides cell treatment, but extended the “glucose clampperiod” of artificially restored normoglycemia from 40 to 120 days. Therationale for this experiment was to give the less efficient irradiateddonor splenocyte treatment an opportunity for more complete hostpancreatic rescue and a regeneration of the pancreatic islet. A cohortof 25 severely diabetic NOD mice was randomized for the same treatmentprotocol of live or irradiated intravenous MHC class I and self-peptideexpressing cells for 40 days with a glucose clamp now implanted for 120days. All treated NOD cohorts of both groups were followed for extendedobservation times for endogenous pancreatic function after clampremoval. Because a glucose clamp containing alginate encapsulated isletshas a greater than 50% failure rate in an autoimmune host after 40 daysof implantation, the glucose clamps used for these diabetic hosts weresyngeneic islets transplanted under the kidney capsule. We removed thisglucose clamp by nephrectomy 120 days after implantation.

Placement of the 120-day extended glucose clamp yielded a markedbeneficial effect when combined with the formerly metabolicallyineffective therapy of irradiated MHC class I and self-peptideexpressing cells followed by an early evaluation at day 40.Functionally, 11 of the 12 NOD hosts which received live MHC class I andself-peptide expressing splenocytes continued to remain normoglycemicfor observation times extending beyond 180 to 250 days after removal ofthe 120 day glucose clamp. Eleven out of the thirteen NOD hosts receivedirradiated MHC class I and peptide expressing cells and remainednormoglycemic for a similar observation time. Both NOD treatment groups,receiving either live or irradiated MHC class I and self-peptideexpressing cells, had physiologically equivalent pancreatic isletdensity as assayed using histological sections of the pancreas. Thepancreatic islets in all cohorts of these two latter groups wereexamined after long-term follow-up of reversed disease for 180-250 daysafter restored normoglycemia. Formerly diabetic NOD cohorts withnormoglycemia after irradiated donor lymphoid injections-possessedhealthy and abundant pancreatic islets, but they were consistentlyaccompanied by impressive circumferential lymphoid infiltrates. Thishistological feature was absent in treated NOD cohorts receiving liveMHC class I and self-peptide donor cells. Therefore, although irradiatedMHC class I and self-peptide expressing donor cells were effective atrestoring long-term normoglycemia due to pancreatic insulin secretion,apparent non-progressive circumferential insulinitis was evident duringlong-term follow-up of the pancreas. Despite the histologicaldifferences, both experimental NOD treatment groups (all 25 cohorts)have pancreatic islets free of invasive insulinitis confirming theabsence of active disease.

We also analyzed these cohorts to identify the actual composition of theinsulin secreting beta cells in the pancreas, the blood, and thesplenocytes at the time of autopsy to determine the contribution of thedonor cells to long-term chimerism or conversion to pancreatic betacells. These initial experiments comparing live to irradiated cellssuggested that live cells had some advantages over irradiated cells.First, the live cells corrected diabetes at a much more rapid rate thanirradiated cells. This result suggested that in the case of a long-termdiabetic in which the regenerative capacity of the pancreas was possiblyless evident, the introduction of live cells that have the ability toconvert to beta cells might be an advantage. On the other hand,irradiated cells, which do not have the ability to regenerate intopancreatic islets, may have an advantage in a new onset diabetic becauseof greater safety. Also, islets in mice treated with live versus deadcells appeared histologically different—in that mice treated with livecells had significantly less circumferential insulinitis than micetreated with irradiated cells which, in many cases, had very impressivecircumferential insulinitis. However, we have no reason to believe thatcircumferential insulinitis eventually progresses to active diseasefollowing treatment with irradiated cells.

There may be at least one more advantage to the regenerating tissuebeing of donor origin. In many of the treated NOD mice, syngeneic islettransplantation was performed under the kidney capsule. In all cases,even after complete disease reversal, syngeneic transplants exhibitedpronounced peri-islet insulitis, in contrast to the regeneratedsemi-allogeneic islets in the pancreas, which were almost entirely freeof peri-insulitis. These data suggest that syngeneic islets themselveshave some yet unidentified defect that promotes an abnormal immuneresponse, even in the presence of a fully re-educated lymphoid system.

Example 4 Reintroduction of MHC Class I Complexes to the Cell forRestored T-Cell Education

The histological impact of the introduction of cells expressing matchedand mismatched MHC class I peptide complexes on treatment outcome wasalso analyzed. As is described herein, treatment with a TNF-alphainducing agent, e.g., CFA, and with cells that express MHC class Ipeptide complexes either on the parenchymal or lymphoid cells resultedin disease reversal in several severely diabetic NOD mice. In contrast,immunization with cells expressing MHC class II peptide complexes wasnot obligatory.

We have demonstrated the therapeutic effectiveness of MHC class Iexpressing islets or splenocytes from C57BL/6 strain carrying theH2K^(b) and H2D^(b) alleles (a matched and a mismatched MHC class Iallele) and a self-peptide complex. We investigated whether thetherapeutic effect was restricted to the matched or the mismatched MHCclass I and peptide molecule or whether the effect had allelicspecificity requirements for stable autoimmune disease reversal. The NODmouse possesses two different MHC class I genes carrying the H2K^(d) andH2D^(b) allele, and the NOD mouse lymphoid cells fail to express anormal density of either self-peptide MHC class I structures. Severelydiabetic NOD mice were treated with CFA in combination with a glucoseclamp of intraperitoneally placed islets encapsulated in alginate orsubrenally transplanted syngeneic islets. The diabetic NOD hostsreceived concurrent biweekly immunizations with parenchymal cell linesexpressing fully NOD compatible MHC class I complexes on fibroblasts(e.g., fibroblast cells from H-2 MHC recombinant donor cell lines) orfully MHC incompatible MHC class I complexes on fibroblasts. All cohortswere sacrificed approximately 40-45 days after treatment initiation forthe histological evaluation of the pancreas. Since the fibroblast celllines represent tumor cell lines, the cells used in these experimentswere irradiated prior to intravenous immunization or injection.

The histological results in the endogenous pancreatic islets differed inthe NOD cohorts receiving fully MHC class I matched or mismatchedpeptide complexes. NOD cohorts randomized to receive MHC class I andself-peptide mismatched cells (H2K^(k)D^(k)), possess pancreatic isletswith massive invasive lymphoid infiltrates in five of the five NOD mice.Not only was invasive insulitis present throughout the pancreas, butnone of cohorts demonstrated a single islet structure withoutinsulinitis or islets with exclusively circumferential lymphoidaccumulation. The histological result obtained with diabetic NOD cohortstreated with intravenous injections of matched donor fibroblasts H2K^(d)and H2D^(d) class I and self-peptide structures were dramaticallydifferent. Treatment with MHC class I matched cells eliminated theinvasive insulinitis in all 14 of the 14 NOD cohorts in the pancreaticislets; 2 of the 14 treated NOD cohorts had pancreatic islets totallydevoid of any invasive or circumferential insulinitis, and 12 of the 14treated NOD cohorts possessed pancreatic islets with mild-to-moderatecircumferential infiltrates.

As presented above, the use of irradiated donor cells frequently resultsin the reappearance of pancreatic islets, but consistently these isletsregardless of the cellular source were accompanied by circumferentiallymphoid infiltrates adjacent to, but not invading, the islet structure.The ability of matched H2K^(d) D^(b) fibroblasts to histologicallyeliminate active islet-directed autoimmunity defined by invasiveinsulinitis confirmed the therapeutic effect of C57BL/6 expressing cellswith a nonspecific effect of the allogeneic H2K^(b) locus, but likelyalso benefited from the MHC class I reintroduction due to the matchedH2D^(b) locus through specific T-cell receptor or cell surface receptor(e.g., CD3) engagement of the host. Therefore, the target of the therapyis likely the host T-cells. Furthermore, non-irradiated F1 splenocytesfrom BALB/C and C57 crosses (i.e., CB6F1/J cells) similarly restoredlong-term normoglycemia in about 75-80% of the formerly diabetic NODhosts receiving a single dose of CFA. Irradiated, mismatched MHC class Iand self-peptide expressing cells were uniformly ineffective inreversing established diabetes. Therefore, this cellular therapyadministered to established autoimmune NOD cohorts had (a) ademonstration that a therapeutic effect can be achieved with only asingle MHC class I allelic match; (b) a demonstration that the cells canbe administered in the presence or absence of allogeneic MHC class I andself-peptide structures; (c) a demonstration that the cells injectedinto diabetic NOD hosts can be live or irradiated donor cells; (d) ademonstration that the cells can be parenchymal or lymphoid in origin;and (e) a demonstration that the cellular effect can be independent ofdonor MHC class I donor cells expressing MHC class II and self-peptidecomplexes since fibroblasts, an MHC class II negative cell type, andlymphocytes showed equal efficacy.

We also performed a cytotoxic T-lymphocyte assay to define the separatetherapeutic effects of MHC class I and self-peptide and the therapeuticeffects of CFA/TNF-alpha for reversal of islet-directed auto-reactivity.As detailed above, an effective therapy to reverse establishedautoimmunity included the introduction of both MHC class I andself-peptide matched cells and administration of CFA or anotherTNF-alpha-inducing agent. Here, the introduced MHC class I andself-peptide complexes were proposed to reselect poorly trained naïvecells with the potential for autoreactivity. Indeed, peripheraltolerance homeostasis appears to be maintained by peripheral MHC class Iand self-peptide complexes, and this prevents naïve cell abundance orunstimulated T-cell abundance—a feature seen in the pre-diabetic NODmouse or in the untreated NOD mouse after the onset of hyperglycemia. Incontrast, the data presented herein indicate that autoreactive memoryT-cells or cells with exposure to antigen stimulated cells areselectively sensitive to CFA presumably due to the obligatory endogenousTNF-alpha induction and subsequent apoptosis due to defects in NFκBsignaling.

To show that these observations could not simply be explained by changesin cell number, we looked at the overall abundance of CD45, CD62L, orCD95. The specific functional role of naïve CD45RB high density cells(CD45RB^(high)) in memory and the role of CD45RB low density(CD45RB^(low)) NOD T-cells in autoreactivity was tested in vitro incytotoxic T-assays to islet targets. While we refer to naïve cells asCD45RB^(high) and memory cells as CD45RB^(low), these cells probably donot represent naïve versus memory cells, but rather represent cells indifferent stages of activation depending on exposure to antigen. Forbrevity, we refer to these cells as mostly stimulated or unstimulatedcells, but sometimes we also refer to these cells as naïve or memorycells. The splenocyte donors, the source of the CTLs, were untreated NODhosts, NOD hosts treated solely with CFA, and NOD hosts treated withboth MHC class I and self-peptide and CFA with long-term diseasereversal. Dispersed NOD islets from 8 week-old NOD female donors wereused as responder cells. We used an insulin enzyme ELISA to detectT-cell lysis of syngeneic islets with insulin release from the target,as well as colorimetric quantitation of insulin by a spectrophotometer.The receptor T-cells were sorted into two pools prior to the assay:unstimulated T-cells defined as CD3 positive, CD45RBhigh, and stimulatedT-cells defined as CD3 positive, CD45RB^(low). Numerous effector totarget cell ratios were tested. Based on these data, an optimized T-celleffector to islet target ratio resulted in co-incubation assays of24-hours of culture at 37° C. Using the colorimetric readout as well asthe direct insulin readout we determined the relative amounts of insulinreleased from live beta cells, and using the ELISA assay we determinedthe actual amount of released insulin in the culture supernatants. Theseresults showed that diabetic NOD derived CD3 cells, either of the memorytype or stimulated type or of the unstimulated type, equivalently lyseddispersed islet cells after 24 hours of co-culture.

Both the colorimetric assay and the actual measurements of releasedinsulin confirmed the pathogenicity of the diabetic NOD cell populationsas showing self-reactivity to syngeneic islet cells. The pathogenicityof stimulated CD45RB^(low) T-cells can be selectively altered. Indeed,splenocytes from NOD cohorts treated with CFA alone 25 days prior to theassay showed the selective elimination of autoreactivity of only thestimulated cell population which has the ability to lyse syngeneicdispersed islet cells. CFA treated NOD cohorts maintained unstimulatedCD45RB^(high) T-cell populations with islet autoreactivity equivalent tothat seen in untreated NOD splenocyte donors. Given that CFA therapyalone, with its resultant endogenous induction of TNF-alpha, was notsuccessful in a diabetic NOD mouse in eliminating existing and latestage autoimmunity disease, the CTL results were consistent with theidea that two identifiable subpopulations of autoreactive cells may needto be manipulated in vivo for disease reversal. A marked contrast isseen in separated subpopulations of unstimulated CD45RB^(high) andstimulated CD45RB^(low) cells obtained from successfully treated NODcohorts that received both syngeneic matched MHC Class I self-peptideexpressing cells and CFA. These mice show complete and stable long-termelimination of both stimulated and unstimulated autoreactive T-cellswith syngeneic islet directed autoreactivity.

Taken together, the results of the CTL assay indicate that in diabeticNOD hosts, unstimulated T-cells identifiable with CD45RBhigh andstimulated T-cells expressing CD45RB^(10W) have islet cytotoxicity orthe potential for islet cytotoxicity. Autoreactive T-cell memorysubpopulations were selectively eliminated with CFA alone, while bothautoreactive stimulated and unstimulated subpopulations were eliminatedwith syngeneic MHC class I and self-peptide and CFA. Both aspects of thetreatment may be required for the elimination of existing autoreactivitydue to the existence of both stimulated and unstimulated cells withautoreactive potential. Accordingly, select treatments may be designedto target and eliminate the separate cell populations. Indeed thishypothesis was supported by our earlier adoptive transfer data showingthat autoreactive NOD cell populations remained after TNF-alphatreatment of diabetic donor splenocytes, presumably because of theability of these cells to change their phenotype and become TNF-alphasensitive after islet exposure.

Example 5 T-Cell Re-Education Due to Exposure to MHC Class I andPresented Self-Peptide

To demonstrate that reversal of established NOD autoimmunity was linkedto MHC class I education of T-cells, we monitored NOD mice before andafter diverse therapies to measure a trend towards restored CD8 T-cellselection. As illustrated in FIGS. 2A-2C, untreated NOD mice or NOD miceonly treated with CFA have high levels of CD62L, CD45RB^(high), and CD95positive CD8 cells. Treatment with CFA and C57BL/6 splenocytes or classII^(−/−) splenocytes decreased the T-cell expression level of CD62L andpartially normalized levels of CD45RB^(high) and CD95 CD8 cells (FIGS.2A-2C). Importantly, the apparent normalization of T-celleducation/selection was not observed in NOD mice treated with CFAtherapy alone (FIGS. 2A-2C). The establishment of normal numbers ofmemory T-cells was not observed when diabetic NOD were treated with CFAand C57BL/6 β₂ m^(−/−) TAP1^(−/−) splenocytes, a cell line with reducedpeptide filled surface class I structures (FIGS. 2A-2C). Long-termmemory requires the surface expression of self major histocompatibilitycomplex molecules, and this positive selection by introduced class Iexpressing splenocytes restores T-cell selection towards normal.

Previously published data supports the concept that CD8 gene expressionis maintained by proper peripheral MHC class I presentation. If class Ieducation is interrupted, treatment of CD8 cells with 0.4% pronasefollowed by 48 hours of culture results in low surface re-expression ofCD8 levels (Pestano, Science 284:1187-1191, 1999). Indeed, C57BL/6splenocytes fully recovered CD8 levels after in vitro pronase treatment:no change in CD8 density was observed after pronase (FIGS. 3A and 3B).In contrast, NOD splenocytes after pronase treatment did not adequatelyre-express CD8 surface levels (FIGS. 3A and 3B). This result confirmspreviously published studies of interrupted MHC class I presentation inthe NOD mouse. Splenocytes from NOD mice, whose diabetes wassuccessfully treated in vivo with C57BL/6 or C57BL/6 class II^(−/−)splenocytes and CFA, had improved CD8 re-synthesis after pronasetreatment in vitro. In contrast, NOD mice treated only with CFA or classI deficient C57BL/6 splenocytes with CFA had persistent problems withCD8 re-synthesis similar to untreated NOD mice, confirming thepersistence of interrupted T-cell selection by MHC class I structure.Simultaneously performed control experiments confirmed splenocytes fromNOD mice of diverse treatment groups and splenocytes from C57BL/6 micere-synthesize CD3 surface proteins at comparable rates (FIGS. 3A and3B). Therefore, four established parameters of interrupted CD8 education(CD45RB^(high) CD62L, cD95, and CD8 resynthesis) due to faulty MHC classI presentation, confirm that NOD mice with disease reversal have partialto complete correction of CD8 phenotypes of T-cell selection.

Example 6 Both Subrenally Transplanted Islets and Endogenous PancreaticIslets Show Equivalent In Situ Islet Regeneration at Both Sites

In the present studies, we demonstrate that an effective therapy canutilize TNF-alpha induction of CFA combined with irradiated or live MHCclass I-matched cells. This combination cures diabetes in over 78% oftreated NOD hosts. In addition, we demonstrate below that functionalpancreatic recovery was slower in the pancreas of cohorts receivingirradiated cells expressing MHC class I self-peptide, although long-termand stable recovery occurs at equal frequency with follow-up in excessof 120 days. To assess the long-term resistance of transplanted isletscompared to re-grown endogenous pancreas islets to disease, additionalsets of diabetic NOD cohorts received either live or dead MHC class Iand peptide expressing splenocytes combined with syngeneic islettransplants, which served as a glucose clamp. The ectopic islettransplants under the renal capsule allowed us to evaluate diseaserecurrence and ectopic islet regeneration compared to that seen in thepancreas. Fluorescence immunocytochemistry was used to compare thepancreatic islets to the subrenally placed islets. In these experiments,we utilized a combination of staining to insulin and BrdU to quantifythe proliferating islet mass at the two sites and to determine apossible difference in resistance of transplanted islets and endogenouspancreatic islets to recurrent disease. We can also quantify possibleproliferation of islet cells and/or their precursors at the two sites intwo successful therapy versions. Since there is speculation thattransplanted islets without their locally adjacent pancreatic precursorcells are end stage cells, these experiments tested the hypothesis thatlong-term islet survival might be an exclusive pancreatic trait. Theselong-term NOD cohorts were compared to severely diabetic NOD mice whichhad received subrenal syngeneic islet transplants 8 days prior to theexperiment, but without the desirable CFA and MHC Class I andself-peptide therapy. Hematoxylin and eosin staining of both subrenaland pancreatic islets of a recently diabetic NOD mouse showed impressiveand large lymphoid infiltrates, almost totally obliterating the newlytransplanted NOD islets, and similarly invasive lymphoid obliteration ofthe pancreatic islets. Moreover, the corresponding inspection of boththe renal and pancreatic sites for insulin positive cells revealed analmost totally negative result. The staining for proliferating cellsassessed by BrdU at both sites also showed the lack of islets in thepancreas and a lack of any proliferation, which suggested that invasiveand autoaggressive insulinitis have recurred or, alternatively, that theongoing disease was not due to local lymphoid proliferation, but rathera migration of these autoaggressive cells to the islet site. In otherwords, the BrdU positive cells were not more highly positive in anactive rejection response, suggesting the active cells migrated to thesite.

As presented above, each pancreatic section showed that NOD hostsreceiving irradiated MHC class I and self-peptide expressing cells havehealthy pancreatic and subrenal islet cells that are surrounded withimpressive circumferential lymphoid infiltrates. These lymphoidaccumulations do not progress to an invasive islet pattern even withlong-term follow-up, nor do they appear to enlarge in the long-term. Theimmunocytochemistry of the islet shows that successfully treated NODmice have insulin positive cells subrenally and in the pancreas.Furthermore, within the islet mass of both the pancreas and subrenalsite of long-term corrected NOD hosts, infrequent but proliferatinginsulin positive cells were observed as demonstrated by the yellow cellsclearly indicating co-staining with insulin and BrdU. Since we used twodifferent dyes to co-stain insulin and BrdU (i.e., red and green,respectively) a co-staining cell is yellow. In addition, based on thereported belief that fully differentiated islet beta cells do notproliferate, and instead are generated from progenitor cells, theinsulin co-staining with BrdU (i.e., the yellow color seen byimmunocytochemistry) likely represents a precursor cell in aproliferative phase. These results demonstrate that long-term endogenousand ectopic subrenal islet survival is possible after the underlyingautoimmunity is reversed. Importantly, in view of our analysis of thisvery late stage after the successful reversal of disease, isletregeneration defined by BrdU and insulin co-staining can occur, althoughat a low frequency, in the pancreas and in subrenally transplantedsyngeneic islets.

Using similar immunohistochemical techniques, we also examined thepancreata from long-term corrected cohorts to determine if the insulinsecreting beta cells in the pancreas were solely due to regeneration ofthe pancreas from endogenous cells or if the regenerated pancreaticislets could also have originated from a donor source (e.g., from theinjected splenocytes). In these experiments, we used formerly diabeticNOD mice cohorts that, after the onset of severe hyperglycemia, (i) weretreated with CFA and fresh F1 splenocytes from male donors administeredin biweekly injections for 40 days, (ii) were implanted with a subrenalsyngeneic islet transplant for 120 days, and (iii) remainednormoglycemic in the long-term when the transplant was removed. Thesemice were subsequently sacrificed at varying time intervals of stablenormoglycemia, usually greater than 60 days. The pancreata of theseanimals were compared to the pancreata of animals that received the sametreatment regimen, except that they received irradiated male donorsplenocytes administered in biweekly injections for the 40-day treatmentperiod. These pancreata were then stained with two-colorimmunofluorescence in which insulin was tagged with a red fluorochromeand a Y chromosomal marker was tagged with a green marker. Allsplenocyte donors were of male origin; therefore this fluorescence assaywas used to determine if any of the insulin positive cells in thepancreas were of male Y chromosome origin. We furthermore performedinsulin co-staining to prove that the Y chromosomes that can be seen inthe islet were of islet origin, and were not of donor lymphoid origin(FIG. 1). Yellow cells indicated the co-staining of insulin and the Ychromosome marker in a single positive cell: yellow cells were only seenin cohorts that received live F1 splenocytes and not seen in thepancreatic islets of cohorts that received irradiated donor stem cells.

Furthermore, the double-positive (yellow) islet cells of donor originwith Y chromosome staining were only seen in the endocrine tissues ofthe pancreas, and not in the exocrine tissues, suggesting that theregeneration had occurred only in the target tissue that was injured.Moreover, in the animals that received irradiated cells, no greenpositive cells (i.e., Y chromosome containing cells) were seen either inthe insulin secreting tissues of the pancreas or in the exocrine tissuesof the pancreas. Furthermore, the cohorts that received irradiated cellsalso never expressed yellow cells in the exocrine or endocrine tissuesof the pancreas, therefore confirming that Y chromosome positive cellswere not present in animals that have received irradiated cells as partof their curative regimen.

Histological analysis of the islets that contained cells of donor originrevealed that, at times, whole islets were of donor origin and at othertimes the peripheral beta cells of the islets were mostly of donororigin. Overall, in a typical pancreas, up to 30% to 50% of the entireislet population of the pancreas appeared to be of donor originsuggesting that this was not an occasional phenomenon of differentiationof blood into islet origin, but was actually quite a dramatic finding.All these immunohistochemical data were derived from cohorts withlong-term normoglycemia, as determined usually around 120 days after theoriginal islet transplant or after the original splenocyte injection.

Experiments performed on NOD mice for the regeneration of pancreaticislets have revealed a number of transcription factors that arebeneficial for the methods of the invention and a number of proteinexpression patterns that are signatures of organ/tissue regeneration.NOD mice have at the site of vigorous islet regeneration increased VEGFexpression, increased Flk-1 expression, and locally high levels ofproteasome function, including high levels of LMP-2 and INF-gamma. Toaccelerate the regeneration process, agents such as TNF-, TNFR agonists,or gamma interferon can be administered to the host prior to theinitiation of regeneration. The administration of cytokines that induceTNF-expression, IL-1 expression, HAT, NF-B, AP-2, EGF-1, Sp1, AP-1,GATA, PECAM-1, activator protein-2, CT-rich Sp1 binding activity,PDGF-A, PDGF-B, monocyte chemoattractant protein-1, TF, Ets1, SCL/Tal-1,FGF, HATs P/CAF, PDGF, CBP/p300 and HIF-2-alpha (HRF, EPAS, HLF) canalso be useful for the acceleration of islet regeneration. In certaincases, islet regeneration can be aided by the administration of VEGF,VEGF fragments, FGF, IGF-1, or by BV endothelium differentiation ortissue regrowth.

In other cases, one or more death receptors (e.g., the death receptorslisted in FIG. 5) are inactivated on the donor cells or one or moreintracellular signaling proteins that mediate cell death are inactivatedin the donor cell to prevent death of the transplanted cells. Forexample, FLIP can be used to down regulate Fas/FasL expression. In otherembodiments, extracellular inhibition or reduction in IL2 (e.g.,inhibition due to chemicals or antibodies) is used to upregulate FLIPwhich then down regulates FAS. In other embodiments, the donor cellshave a blockage of IL2R, such as the binding of a chemical (e.g., anon-lytic antibody fragment) to IL2R to inhibit binding of IL2 to IL2Rand thus IL2-mediated upregulation of FAS. In other embodiments, one ormore members of the intracellular pathway for FAS activation areinhibited in the donor cells prior to transfer. Examples include theinhibition of the translation of transcription factors such as cFOS,cJAN, PKC, Lck, Zap70, MAPK, Itk (IL-2 inducible T cell kinase) and JNK.In particular embodiments, the transcription or translation oftranscription factors is transiently inhibited with antisenseoligonucleotides or by RNA interference (RNAi).

Promotion of islet regeneration can be accomplished using one agent, ormore than one agent, administered with or without pluripotent cells. Theprogress of islet regeneration can be monitored using sequential RT-PCRanalysis to probe for the induction or suppression of transcriptionfactors after agent administration.

Example 7 Donor Derived Cells are Also Present in the Blood

Because of our dramatic findings in the pancreas of donor origin F1cells turning into pancreatic islets, we also serially examined both theblood and splenocytes from these cohorts to see if the blood andsplenocytes were also of donor origin. Approximately eight cohorts ofthis long-term description were examined for the presence of K^(b)positive lymphoid cells in the peripheral blood; splenocytes at the timeof sacrifice were also examined. As is noted above, K^(b) cells must beof B6 origin because the NOD mouse is of K^(d) origin. We analyzedperipheral blood lymphocytes from these cohorts using flow cytometryanalysis and found that in the peripheral blood 12.6%, 8.3%, 10%, 0.9%,4.4%, and 5.8% of the lymphocytes were of donor origin. In contrast, acohort that received irradiated cells, in which staining would onlyrepresent endogenous staining (i.e., background staining), had 2.9% oflymphocytes of donor cell origin. Thus, many of these cohorts had apercentage of donor origin lymphocytes in the peripheral blood that wassignificantly above background and had long term co-existence of bloodcells of two different genetic origins and pancreas cells of twodistinct genetic origins.

To better define this co-existence of donor derived and endogenous cellswithout immunosuppression, skin transplants were also performed on theselong-term cohorts from the B6 donor. We had presumed that since therewas blood chimerism and now pancreatic chimerism, the skin graft wouldsurvive long-term. To our surprise, skin graft survival from the B6cohorts was not prolonged, or not visibly prolonged, in cohorts thatretain stable blood and pancreatic islet chimerism, indicating that thissort of chimerism is distinct from the chimerism that results from totalbody irradiation followed by bone marrow reconstitution.

Nonetheless, the methods described herein provide a remarkable way totransplant cells without the need for immunosuppression. In view of thestandard knowledge in the field of transplantation prior to the presentinvention, donor cells that not only are chimeric—being of donor maleorigin bearing disparate MHC genes and remarkably turning intopancreatic islets—but also are semi-allogeneic would be expected to berejected because, while the host received CFA or TNF-alpha, the host didnot receive immunosuppressive treatment. However, as is shown by ourresults, we were able to maintain long-term chimerism. In many ways thestable chimerism that could persist beyond 180 days after therapytermination mimics pregnancy where fetal origin F1 cells can survivelong-term in mothers, long after the fetus has been removed.

Example 8 Organ Regeneration in GFP C57BL/6 Mice

As noted above, the data described herein using mice with establisheddiabetes (e.g., NOD mice) demonstrate the ability to re-grow islet cellsin the pancreas. The experimental results are excellent and demonstratea robust and sustained ability to achieve engraftment. To try toduplicate these results, and to determine the parameters that allow thisremarkable phenomenon to occur, we set up a test system to define theparameters that allow the NOD mouse to re-grow its islets from donorblood cells. The test model used cells from GFP BL/6 (B6) miceexpressing green fluorescent protein (GFP) in all tissues as donor cellsfor introduction into B6 cohorts. Initially, we used GFP B6 splenocytesinjected into normoglycemic hosts. We then examined these hosts atvarying intervals for pancreatic, lung, and blood chimerism. After 90days, no chimerism of the donor origin was visible. Based on thesefindings, we decided to test the possibility that the host pancreasneeds to have an insult (e.g., the co-administration of streptozotocinto allow the GFP positive B6 donor lymphocyte cells to target thepancreas and also regenerate it). Therefore, GFP positive B6 cells fromsplenocytes and bone marrow (Hoechst 33342/SP positive cells) obtainedby flow cytometry and hepatocyte origin cells were administered at dosesof 5×10⁵ to 5×10⁷ cells over a 40-day period, and the cohorts were thenexamined after 40 to 195 days either by eye bleeds or by sacrificefollowed by examination of splenocytes.

In these experiments, although there was injury to the pancreas, therewas little persistence of long-term chimerism in the host animals.Occasionally, a pancreas positive cell of GFP origin was observed, butthe data were in large part negative suggesting that we had not properlyduplicated the experiments that were so successful in the NOD mouse. Onepotential reason for the lack of success of this experiment or for thesuccess of the experiments in NOD mice is that although we had inducedinjury in the pancreatic islets, these animals were severelyhyperglycemic. Based on our previous data, severe hyperglycemia hamperedregeneration.

To determine if severe hyperglycemia was interfering with theregeneration of the pancreas, we repeated the experiments usingstreptozotocin induced damage and a glucose clamp with subrenal isletsand then used donor splenocytes or donor bone marrow from GFP positiveB6 donors. In response to this treatment, the chimerism was stillpartial, not long-term, and did not represent the striking regenerationof the islet tissue.

We further optimized the treatment by administering streptozotocin toanother set of B6 cohorts, inducing the glucose clamp with subrenallytransplanted syngeneic islets, and co-administering TNF-alpha or CFAconcurrent with the donor lymphoid cell injection. We used this protocolbecause we thought that we needed injury to islets to result in high TNFReceptor 2 expression on the islets or growth receptors to perhapspromote regeneration of the endogenous pancreatic islets. Furthermore,we thought that TNF Receptor 2 and progenitor cells from the blood mightalso promote endogenous GFP positive B6 islet regeneration and that CFAand TNF-alpha might be beneficial in another manner. We had previouslyobtained data indicating that CFA or TNF-alpha induces severe transientlymphopenia in the host, which is similar to data obtained in humanclinical trials. Therefore, we injected the six hosts, not only withstreptozotocin and an islet transplant, but also with CFA or TNF-alphato induce the severe transient lymphopenia that might promote theperipheral blood chimerism. In addition, we injected splenocytesbiweekly for 40 days. The results of these experiments looked much morepromising, as 2-10% chimerism was detected in the peripheral blood 180days after the completion of the injection and, furthermore, GFPpositive cells of donor origin, although rare, were vividly expressed inthe islets of the pancreas.

In short, in the syngeneic situation, splenocytes differentiated intoinsulin secreting beta cells, fused with beta cells, or provided factorsfor regeneration. We determined that, in the C57BL/6 host, CFA orTNF-alpha is desirably not administered concurrently with the donorcells. Therefore, these experiments using syngeneic transplants insteadof allogeneic transplants and using an artificial model of islet injurysuggest that target organ injury or active disease promotes theregenerative process after the elimination of the disease. Ametabolically normal state is also important and may need to bemaintained, as severe hyperglycemia appeared to interfere with theeffectiveness of this treatment. Our results also indicate thatTNF-alpha or CFA may facilitate the effectiveness of the treatment.These results likely represent a dual effect, not only of CFA'selimination of autoimmunity in the NOD mouse, but also of CFA'sinduction of severe lymphopenia, which, in turn may promote thechimerism of donor cells, as well as the subsequent chimerism anddifferentiation in the pancreas. Also, induction or administration ofTNF-alpha has a beneficial effect on the target tissue or precursorcells promoting regeneration. Furthermore, it is known that the bestinduction of host regeneration, based on percentage success rate (92%vs. 72%), as well as the percent degree of chimerism/regeneration(approx. 87% vs. 54%), is still obtained from the administration of CFA,which is somewhat superior to the administration of TNF-alpha alone. Itshould be noted that once animals are successfully treated with eitheragent, the stability of disease reversal is equivalent. Although theseresults could be due to dose response phenomena, it is also observedthat the simultaneous induction of INF-gamma with CFA is of directbenefit in conditioning the host vascular endothelium for recapitulatinga regenerative program. Indeed, INF-gamma induces both high LMP2 andother proteasome subunits that promote vascular leakiness, a necessarystep to presumed mesodermal cell migration and differentiation.

While the above experiments relate to the treatment of diabetes, thesetechniques obviously also are applicable to other diseases where hostrepair is desirable, providing new ways to transplant cells without theneed for immunosuppression.

Example 9 Organ Regeneration in GFP C57BL/6 Mice

As noted in Example 7, additional experiments were performed usingnormal mice that are not of the NOD genotype to further understand andcharacterize the remarkable re-growth of islet cells observed in NODmice.

FIG. 9 summarizes the many C57BL/6 mice that were treated with varioustherapies to achieve similar donor cell engraftment and possiblere-growth of an adult organ/organelle such as the islets of Langerhans.All of these C57BL/6 hosts were made diabetic with streptozotocin usingstandard methods. In these experiments, glucose levels were notregulated using insulin injections or a temporary glucose clamp. Ifdesired, insulin injections or a temporary glucose clamp may be used inany of the methods described below to optimized islet cell regeneration.It has been observed by us that in the NOD host, diabetes or late stageislet destruction is necessary for islet regrowth and thus to create asimilar model of injury of the pancreatic islet, injury was induced withstreptozotocin prior to the introduction of donor cells.

The Group 1 mouse was a female C57BL/6 mouse that received donor malesplenocytes from a syngeneic C57BL/6 donor with GFP-actin fluorescence(C57BL/6-GFP). Thus, the donor cells can be distinguished fromendogenous cells because the donor cells are of male origin (i.e., haveXY chromosomes) and the endogenous cells are of female origin (i.e.,have XX chromosomes). Additionally, the cells exhibit GFP fluorescencethat is easily detectable using flow cytometric analysis. Removal ofblood from this host and analysis of PBLs revealed that the peripheralblood only had 0.59% GFP cells. This demonstrates that the introductionof cells of splenocyte origin was not sufficient for establishing highlevels of chimerism under the conditions tested. Also, the 0.59% valuerepresents the degree of chimerism approximately four days after thefinal bi-weekly injection of donor splenocytes expressing GFP suggestinglow levels of C57BL/6-GFP cells remained.

Group 2 C57BL/6 mice were treated as described for the Group 1 host. Thespleen and the PBL of the treated mice were analyzed 150 days aftertreatment began. This treatment regimen involved bi-weekly injections of10⁷ cells for the first 40 days after treatment. The PBLs of the group 2hosts also had low levels of donor cells. The spleen had slightly higherbut still low levels of C57BL/6-GFP cells. A subset analysis seemed tosuggest the blood cells expressing GFP were not confined to any selectlineage. Group 2 910, 911, and 903 NOD hosts were reanalyzed by regatingthe flow pictures of fluorescence and had similar trends of low levelsof chimerism.

Group 2 mice 931, 939, 932, and 933 were also studied 87-98 days aftertransplantation of C57BL/6 splenocytes. The spleen of these animals hadslightly higher degrees of chimerism with ranges of 4.7-13.3%. Althoughthis result suggests a detectable level of chimerism, this chimerism wasnot long lasting because by an additional 100 days, the degree ofchimerism was again low.

Group 3 and 4 C57BL/6 mice only differed from Group 2 and 3 mice in thatGroup 3 and 4 mice received donor bone marrow cells instead ofsplenocytes. Splenocytes were better able to engraft into the host thanbone marrow donor cells. Group 5 C57BL/6 hosts received Hoechst333242positive splenocytes; a cell type that is alleged by the scientificliterature to poses stem cell traits. The transfer of these cells intothe C57BL/6 hosts was only minimally successful and less successful thandonor bone marrow or bone splenocytes.

Lastly, Group 7 and Group 9 C57BL/6 hosts received 10⁷ CNS precursorcells or hepatocytes (HC), and the hosts were killed approximately 100days after cell transplantation. The spleen of hosts demonstrated moreengraftment than the PBL, and donor CNS cells or donor hepatocytes maybe better able to engraft into the host than bone marrow orHoecchst33342 cells.

It should be noted for all these experiments in all groups we never sawwith donor cell treatment reversal of the diabetes and we did notobserve above background levels a clear regeneration of the islets inthe pancreas. The pancreases possible regrowth, even temporary, wouldprobably not have been detected with this experimental design becausethe killing of the mice was late in most cases. Future experiments werethus conducted to see if like the NOD a simultaneous tight control ofblood sugars was necessary to promote islet regrowth during theexperimental observation period and to perpetuate the chimerism in atarget organ.

In the NOD model of disease reversal and islet re-growth, the data showthat diabetic NOD mice that receive both CFA and donor F1 splenocytesexhibit islet regeneration (FIG. 10). A glucose clamp was used toregulate glucose levels and enhance islet regeneration. The data showthat even very low dose TNF-alpha (e.g., doses of 2 ug/bi-weekly) canalso promote the reversal of disease process. Further experimentsrevealed that the substitution of CFA with TNF-alpha required TNF dosingof 10-20 ug/bi-weekly. The NOD data also clearly shows that CFA alone ordonor splenocytes alone were without long lasting effect at eitherdisease elimination or islet regrowth at the time periods examined.

The second part of FIG. 10 now attempts to translate the NOD successstory of organ regeneration into a C57BL/6 model of regeneration. Thishas helped to define the critical elements that promote regeneration. Inall of the C57BL/6 mice in these groups, streptozotocin was used toinduce tissue injury and to make the mice diabetic. As noted above,tissue injury promotes re-growth. Again, the groups that appear to havetarget organ regeneration are the groups that receive donor splencoytesplus TNF-alpha. In these experiments we try to map the pathway orreceptor for regeneration as involving receptor I or II. At least forreceptor II stimulation with the use of a C57BL/6 mouse with a mutationthat inactivates TNF-α receptor I, we can see the persistence of theislet regeneration to a certain degree suggesting the islet regenerationmay be promoted by this later pathway. The complete experiment could notbe done in the reciprocal fashion because even very low dose TNF-alphaadministered to a C57BL/6 RII−/− mouse resulted in immediate death;TNF-alpha toxicity may be through this receptor I, at least in controlmice.

The last portion of FIG. 10 examines the effect of TNF in NOD mice intransiently promoting islet regrowth and the rapid elimination ofinvasive insulitis. With escalating doses of TNF, one can see not onlythe elimination of invasive insulitis but also islet regeneration. TheseNOD cohorts were typically examined about 40-50 days at the end of theTNF treatment course. Based on examinations of histological sections forTNFR11 expression, regenerating islets demonstrated up-regulation ofthis receptor while there was still some tissue injury. Thisup-regulation of TNFR11 may promote the beneficial effect of TNF inregeneration. For example, 20 ug dosing of TNF eliminated all theinsulitis and resulted in regeneration of the islets to the mostsignificant degree. Examination of NOD mice being treated with 20 ug TNFat earlier times prior to the end of the 40-day period would like revealhigh TNFR11 expression that is eliminated by the end of the 40-dayperiod because islet regeneration is complete. Also, treatment of NODmice with human-TNF-, an agonist of only TNFR1 in the mouse, resulted inno islet regeneration, suggesting the beneficial effect of TNF- on organregeneration was a function of the effect of TNF- as an agonist ofTNFRII.

FIG. 11 summarizes the diverse experiments and outcomes depending uponthe host representing an NOD mouse or a normal C57BL/6 host. The use ofcells of splenocyte origin, blood origin or HC may offer an advantagebecause these organs contain diverse cell types and the re-introductionof mobilized, but not yet fully differentiated, endothelium, mesoderm,or ectoderm may promote, facilitate, or speed the necessaryrecapitulation of fetal tissue interactions that promote organregeneration in an adult. The following data support the abovehypothesis. During normal embryonic pancreatic islet development, themesoderm interacts with the BV endothelium (endoderm). This interactionmay promote VEGF expression, as well as the upregulation of Flk-1receptors. To promote this process of organ specific regeneration in anadult, a number of steps are desirably followed. First, cells of theoriginal developmental contact are desirably administered by IVinjections or applied directly to the site of regeneration. Forregenerating islet cells, blood vessel (BV) endothelium is desirablyprimed at the regeneration site by promoting the embryonic expression ofVEFG, NF-B or associated events, such as increased proteasome activityor TNFR2 expression, and then contacted with administered mesodermalcells, even if of adult origin. For example, injected mesodermal cellsmay contact endogenous endoderm (e.g., endodermal cells within thepancreas or within other areas of the body), which promotes therecapitulation of the fetal patterning, i.e. the BV endothelium plusendoderm budding produces islets of liver cells. Indeed, in thisparticular clinical setting, the power of donor splenocyte origin cellsin promoting regeneration may be more attributable to the mesodermalcells of the spleen than the more abundant blood cells. For re-growth ofother tissues, administration of ectoderm, mesoderm, and/or endoderm maybe desirable. Furthermore, for target organ re-growth, transientup-regulation of VEGF may be desirable. This up-regulation may beinduced, e.g., by administering TNF-alpha, INF-gamma, or inhibitingcAMP. Also, administration of IL-2 may promote TNF-alpha thatsubsequently binds to BV endothelium, triggering VEGF up-regulation andNF B up-regulation, and thus target organ regeneration. Since TNFR2 ispreferentially expressed on endothelial cells, this receptor isdesirably manipulated for target organ regeneration. The ability of theNOD mouse to regenerate islets as demonstrated herein may beattributable, at least in part, to the islet specific up-regulation ofthe LMP2 subunits of the proteasome. Up-regulation of LMP2 is veryinfluential in promoting VEGF/Flk-1/TNF-effects, with NF-B upregulation,as we now demonstrate by its diminished effect in LMP2−/− mice. We havedemonstrated this regenerative process to be promoted in the NOD mouseand eliminated in the LMP2−/− mouse, thus verifying this pathway.

If desired any of the above regeneration methods may be enhanced byadministering the donor cells more frequently and/or for a longer lengthof time.

Example 10 Assay Development of Human Diabetic Peripheral BloodLymphocytes

As the relative efficiency of donor NOD splenocytes in transferringautoimmune disease is well known and NOD blood is very inefficient as asource of lymphoid cells in transferring disease to naïve cohorts, themagnitude of apoptosis induced by TNF-α in pathogenic NOD T cells fromperipheral blood compared with the effect in T-cells from NODsplenocytes was quantified.

As Table 4 shows, accelerated cell death in NOD splenocytes, measured asboth early and late apoptosis, resulted in 46% cell death. The effect onperipheral blood lymphocytes (PBLs) in the same NOD mouse was only 12%induced apoptosis. The distribution of pathogenic apoptotic sensitivecells appears to be lower in peripheral blood and higher in the spleen.

TABLE 4 TNF- sensitivity of PBLs vs. splenocytes in NOD mice* Apoptosisof T Cells (%) TNF- Spleen PBLs  0 ng/mL 12.1 12.1 12.2 15.6 20 ng/mL11.8 22.6 11.5 17.7 *Apoptosis of T cells represents early and lateapoptosis defined as Annexin V+ PI+ and Annexin V+ PI− cells on CD3+ Tcells using flow cytometric studies

The data in Table 5 show the degree of accelerated T cell death of humandiabetic PBLs with culture and with TNF-α, as measured by flowcytometry. Apoptosis was quantified by flow cytometric monitoring ofAnnexin V, with or without propidium iodine staining, after a 12 hour invitro culture or exposure to TNF-(20 ng/mL), TNF-with Act D (1 ng/mL),or other protein synthesis inhibitors known to amplify pro-apoptoticpathways of TNF-signaling by inhibiting the rapid synthesis of proteinsthat are anti-apoptotic. All assays were performed on freshly isolatedPBLs and simultaneously prepared control samples. Both early and lateapoptosis results were pooled for these data, but early and lateapoptosis each was sufficient by itself in each category in Type Idiabetics to yield highly statistically significant values ofaccelerated death through culture with TNF-. With flow cytometric data,profound changes in the relative mean death can be observed on any givenday, so patient samples were always simultaneously studied and comparedto paired t tests to control samples. The magnitude of the TNF-α inducedapoptotic defect in humans is detectable with current flow cytometrytechniques (8-10%) and is consistent with the results in PBLs in the NODmouse. The 55 type 1 diabetic patients had higher death of naïve T cells(with culture) compared to 55 paired random (no history or familyhistory of autoimmune disease) controls (p=0.0008). Actinomycin D is anaccelerator of apoptosis when used with TNF-α. As shown in FIG. 5, TNF-αand TNF-α plus actinomycin D (p=0.0007) induced apoptosis were alsosignificantly greater in the human diabetic T cells than in the controlT cells (p=0.0154 and p=0.0007, respectively). The data suggest that thedefect is widespread in Type 1 diabetes, with the majority of patientsshowing a detectable abnormality in T-cells (with a relatively largerfraction of T-cells with heightened TNF-α sensitivity).

It should also be mentioned that, similar to the NOD mouse, thereappears to be two death-mediated events, a spontaneous death of cellswith tissue culture preparation and a direct TNF-induced death of selectT cell subpopulations. The spontaneous cell death maps to themonocyte/macrophage lineage of cells and the direct TNF-death maps inboth species of T cells. The spontaneous death could be due to receptoractivation of a death receptor due to shear stress or, alternatively,the elimination in the autoimmune patient of an abnormal serum factorthat is abnormally pro-life or anti-apoptotic.

TABLE 5 TNF-induced apoptosis of peripheral blood lymphocytes of Type Iand Type II diabetics compared to controls Com- Paired Mean Mean %Paired t parison Samples Conditions (patient) (control) Change test TypeI 55 Culture - 28.8 26 10% 0.0008 diabetic 12 hrs vs. Control Type I 55TNF 29.6 27.2 8% 0.0154 diabetic vs. Control Type I 55 TNF + 42.8 39.210% 0.0007 diabetic Actinomycin vs. D Control Type II 18 TNF 26.5 26.91% 0.9422 diabetic vs. Control Type II TNF + 38.8 38.1 −2% 0.5702diabetic Actinomycin vs. D Control

Example 11 Treatment, Stabilization, or Prevention of Disease Other thanDiabetes

NOD mice also suffer from other autoimmune diseases in addition todiabetes, such as rheumatoid arthritis, lupus, multiple sclerosis,Sjögren's syndrome, multiple sclerosis, and autoimmune hemolytic anemia.In particular, the methods of the invention also improved symptomsassociated with these other autoimmune disease and/or stoppedprogression of these diseases in NOD mice. The following treatments havebeen tested and shown to enhance regrowth of salvary glands, decreasehemopoetic abnormalities, stop the progression of multiple sclerosis andrheumatoid arthritis, and reduce levels of lupus autoantibodies: (i)biweekly injections (i.v.) of 10⁷ splenocytes expressing MHC class I andpeptide for 40 days, (ii) biweekly injections (i.p.) of 2, 4, 10, or 20μg TNFα or IL-1 for 40 days, (iii) a single injection of 5 μL in onefootpad of 1 mg/mL solution of BCG, (iv) a single injection of CFA, (v)combined treatment with splenocytes and TNFα at the above doses, and(vi) combined treatment of splenocytes and CFA at the above doses.

Mice such as C57BL6 mice can also be used as animal models for theregeneration of other cells, tissues, or organs such as skin, liver, orbrain cells.

Example 12 Factors Affecting the Efficiency of Organ Regeneration

Our data using GFP mice also demonstrated that, as we repeated theseexperiments with many different types of injected donor cells,differences exist not only in the degree of peripheral blood chimerism,but also in the persistence of peripheral blood chimerism induced bythese different donor cells. As is noted above, the GFP positive donorcells that we obtained and injected included splenocytes, bone marrowderived cells, Hoechst 33342 positive cells obtained by cell-sorting,brain cells, CNS derived cells, and hepatocytes. Based on analysis ofperipheral blood lymphocytes after sacrificing the animals for analysisof splenocytes, the duration of the chimerism in the absence of CFAtreatment was dramatically different for different cell types. It turnsout that, of the different cell types tested, splenocytes maintained thehighest degree of chimerism for time periods greater than 100 days. Incomparison, donor bone marrow cells were less effective, and the othercell types were least effective, suggesting that the donor cell origineven from the adult donors may also have an impact in the persistence ofthe chimerism.

In autoimmune hosts, the administration of any of a multitude ofcytokines induce death or apoptosis of a subpopulation of pathologiclymphoid cells due to these cells having intrinsic errors in resistanceto apoptosis or cell death. Accordingly, this treatment eliminates thepathologic cells from the host without harming the endogenous cells. Inaddition, introduced and endogenous cytokines promote the regenerationprocess of a damaged target organ. If a target organ is inflamed, isexposed to exogenous cytokines, or has increased proteasome activity,such as increased activity due to the overexpression of the LMP2, LMP7,or LMP10 subunits, a gamma responsive gene, or a TNF-alpha responsivegene, the target organ regenerates at an exponential rate. An increasein proteasome activity is likely to play a role in the action of VEGF,which together with the VEGF receptors Flk and Flt, functions in organregeneration. Studies have shown that VEGF binds to developing organsand that this promotes end organ regeneration, possibly by binding toFlk or Flt receptors. We have shown that augmented proteasome activityresults in augmented VEGF activity. In view of these results, in anautoimmune host, it is likely that, once disease is removed and in situproliferation is desired, stem cells home to the target organ that hadbeen under autoimmune attack and preferentially proliferate in thisorgan. The upregulation of proteasome activity and/or the upregulationof proteasome subunits with gamma interferon may promote this. Inaddition, gamma interferon may be used in a non-autoimmune host withtissue damage to promote targeting of this damaged tissue by stem cells.Furthermore, other chemicals and cytokines that also promote proteasomeactivity may be used in methods of organ regeneration. For example, insuch methods, a promoter of proteasome activity may be administeredconcurrently with, prior to, or after administering stem cells orlymphocytes obtained from adult blood. After the addition of stem cells,local regeneration may be promoted by increasing Flk-1 receptors viaCREB inhibition or by TNF-, HAT or NF B activation, or by administrationof VEGF inhibitors. VEGF secretion may be promoted by proteasomeaugmentation, TNF-administration, cAMP inhibition, by the administrationof IL-1 or IL-2, or by the application of sheer stress.

Example 13 Exemplary Agents for Use in the Methods of the PresentInvention

Select autoimmune cell death can be achieved by administering agentsthat disrupt the pathways that normally protect autoimmune cells fromcell death, including soluble forms of antigen receptors such as CD28 onautoreactive T-cells, CD40 on B-cells that are involved in protection ofautoimmune cells, and CD95 or CD95L (i.e., FasL) on T-lymphocytes. Othersuch agents include p75N TNF and lymphotoxin Beta receptor (LtbetaR).Also, antibodies or fragments of antibodies reactive with thesereceptors are useful therapeutics. Such agents are described in theliterature.

The present invention is not limited to a combined TNF-inducing therapyor direct compound administration that includes the combination ofTNF-alpha and IL-1, but includes, e.g., any combination ofTNF-alpha-including therapies, e.g., vaccination with BCG, viralinfection, LPS, activation of cells that normally produce TNF-alpha(i.e., macrophages, B-cells, and T-cells), administration of thechemotactic peptide fMet-Leu-Phe, administration of bacterial and viralproteins that activate NF_(κ)B, administration of agents that inducesignaling pathways involved in adaptive immune responses (i.e., antigenreceptors on B- and T-cells, CD28 on T-cells, CD40 on B-cells), agentsthat stimulate specific autoreactive cell death receptors (i.e., TNF,Fas (CD95), CD40, p75NF, and lymphotoxin Beta-receptor (LtbetaR), andadministration of substances that stimulate TNF-alpha converting enzyme(TACE) which cleaves the TNF-alpha precursor (i.e., to providebiological activity capable of stimulating enhanced production orenhanced cytokine life after secretion). Such agents are described inthe literature.

In a preferred embodiment, monoclonal antibodies that serve asTNF-agonists can be administered. Such antibodies can be made usingtumor necrosis factor-alpha receptor 1 (TNFR1) or tumor necrosisfactor-alpha receptor 2 (TNFR2) as immunogens in mice using thehybridoma method first described by Kohler & Milstein, Nature 256:495(1975). Such antibodies can also be made by recombinant DNA methods[Cabilly, et al., U.S. Pat. No. 4,816,567]. Such antibodies have beenprepared and described by Brockhaus, et al., in Proc. Nat. Acad. Sci.USA 87:3127-31 (1990). Among the antibodies produced, those with agonistactivity are identified by screening for TNF-like activity in assaysmeasuring cytotoxicity, fibroblast growth, interleukin-6 secretion, oractivation of the transcription factor NF-B. Alternatively, suchantibodies can be screened in vitro using assays in which agonists areidentified by their ability to kill activated T-cells obtained, forexample, from a patient with lymphoma or newly diagnosed type-2diabetes.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a non-human source. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. In antibodies used in themethods of the invention, the import variable domain is from the TNFR1and TNFR2 antibodies produced above. Humanization can be performed, forexample, following the method of Winter and co-workers [Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);and Verhoeyen et al., Science 239:1534-1536 (1988)], by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (Cabilly, supra), wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and, in somecases, some FR residues are substituted by residues from analogous sitesin rodent antibodies.

Humanized antibodies desirably retain high affinity for the immunizingantigen, and thus are desirably prepared by known processes involvinganalysis of the parental and humanized sequences by three-dimensionalmodeling. Three dimensional immunoglobulin models are commonly availableand are familiar to those skilled in the art. Computer programs areavailable which illustrate and display probable three-dimensionalconformational structures of selected candidate immunoglobulinsequences. Inspection of these displays permits analysis of the likelyrole of the residues in the functioning of the candidate immunoglobulinsequence, i.e., the analysis of residues that influence the ability ofthe candidate immunoglobulin to bind its antigen. In this way, FRresidues can be selected and combined from the consensus and importsequence so that desired antibody characteristics, such as increasedaffinity for the target antigen(s), are achieved. In general, the CDRresidues are directly and most substantially involved in influencingantigen binding. For further details see U.S. Pat. No. 5,821,337.

Alternatively, it is possible to produce transgenic animals (e.g., mice)that are capable, upon immunization, of producing a full repertoire ofhuman antibodies in the absence of endogenous immunoglobulin production.For example, the homozygous deletion of the antibody heavy chain joiningregion (J_(H)) gene in chimeric and germ-line mutant mice, resulting incomplete inhibition of endogenous antibody production has beendescribed. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA 90:2551-255 (1993); Jakobovits et al., Nature362:255-258 (1993).

In the use of a TNF-receptor agonist antibody, patients are dosed suchthat enough is administered to elicit a TNF-like effect. The effectivedose of such an antibody, or mixture of antibodies, is determined bystarting at a low dose to ascertain tolerance, followed by doseescalation to produce the desired changes in circulating lymphocytes.For example, therapeutic dosing can be weekly or bi-weekly at levels of0.025 mg/kg, 0.075 mg/kg, or 0.150 mg/kg (antibody/patient). Subsequentto antibody administration, disease activity is monitored in eachpatient category. For diabetes, monitoring may involve the tabulation ofthe amounts of insulin necessary to maintain normoglycemia or a positivetrend in the reappearance of C-peptide levels. All patients during andafter monoclonal antibody therapy are monitored for the presence of ahuman anti-murine antibody response to the anti-TNFR antibodies, as wellas a human anti-human response.

In another preferred embodiment, the invention allows for theidentification of drugs that induce cell death or selectively hamper theautoimmune cells by binding to cell surface receptors or interactingwith intracellular proteins. For example, drugs that stimulate the IL-1pathway or drugs that interact with converging pathways such as Fas,FasL, TACI, ATAR, RANK, DR5, DR4, DCR2, DCR1, DR3, TALL-4, or THANK.Also accelerated cell death of autoimmune cells maybe potentiated byadding protein synthesis or kinase inhibitors. For instance, acceleratedTNF or FAS death is potentiated by brief exposure to a protein synthesisinhibitor (e.g., ActD) that blocks a rapidly made TNF-alpha mediatedintercellular inhibitor(s). Similarly, kinase inhibitors also potentateTNF-alpha mediated events. The drugs of the present invention can becharacterized in that they only kill autoimmune cells having a selectivedefect in a cell death pathway which can be characterized by twodistinct phenotypes, (1) defects in lymphoid education and (2)susceptibility to apoptosis.

Other host treatment methods can be used as well to ablate autoimmunecells, for example, administration of CFA, interleukin-1 (IL-1),proteasome inhibitors, TNF superfamily agonists, NFκB inhibitors,anti-inflammatory drugs, tissue plasminogen activator (TPA),lipopolysaccharide, UV light, or an intracellular mediator of theTNF-alpha signaling pathway.

Example 14 Treatment

While the therapies described herein are likely to be effective intreating pre-diabetics, i.e., patients diagnosed as progressing to typeI diabetes, but who are not yet hyperglycemic, we note that the methodsof the inventions also may be used to treat a mammal, for example, ahuman with type I diabetes or any other autoimmune disease. The abilityto treat patients who already have hyperglycemia and therefore havesignificant or total islet destruction is a significant advantage of thecurrent therapy.

In general, before treating a patient, one may optionally obtain bloodfrom the patient to determine that the patient has two diseasephenotypes. The first disease phenotype is an increase in the number ofcirculating CD45RA positive cells in the blood (also defined asalterations in the number of cells positive for CD95, CD62L, or othermarkers of naïve or unstimulated cells). CD45, CD95, and CD62L are allcell surface antigens that can be monitored by flow cytometry andcompared to age matched controls. We expect to see an abundance of thesenaïve or unstimulated cells in the peripheral blood of subjects withdiabetes or any other autoimmune disease. The second phenotype is thepresence of a subpopulation of lymphocytes with augmented sensitivity tocell death through apoptosis or necrosis. For example, subpopulations ofcells may have augmented sensitivity to cell death caused by TNF-alpha,TCR receptor cross-linking agents, T-cell specific antibodies (e.g.,αTCR or αCD3), or nonspecific stimulation with BCG. We may assay for thepresence of such cells by isolating lymphocytes from these patients,treating them in vitro with TNF-alpha, and showing that the lymphocytescontain a subpopulation that undergoes apoptosis or necrosis whenexposed to TNF-alpha, other cytokines, chemical reagents, or antibodiesto select surface proteins. Desirably, control donor lymphocytes do notexhibit sensitivity to these agents. This phenotype is a result oflymphoid cells predominantly of pathogenic origin that have alteredintercellular signaling pathways, alterations which result in aheightened death sensitivity. Elimination or conversion of all cellswith this phenotype is desirable for the permanent reversal ofautoimmunity. The penetrance of these defects is likely to be relativelyhigh in diabetic or other autoimmune patients, with the first phenotypelikely having a penetrance of over 95%, and the second phenotype likelyhaving a penetrance of over 50% in type I diabetics.

Accordingly, before beginning to treat a subject with type I diabetes orany other autoimmune condition, we may determine from blood analysisalone whether the subject has either or both of these two phenotypesand, therefore, is amenable to therapy. To treat the first phenotype(i.e., an increase in the number of circulating CD45RA positive cells)tolerance to MHC class I and self-peptide may have to be re-established.We conclude from our results that the lack of functional MHC class I andself-peptide complexes causes the overabundance of naïve T-cells in theperiphery or at least results in one of the phenotypes that causes this.So for treating this phenotype, we can administer blood or bone marrowthat is a semi-allogeneic or fully-allogeneic match to the MHC class Iand self-peptide complex. Furthermore, the blood or bone marrow derivedcells, or even fibroblasts that have been immortalized, desirably mayhave normal MHC class I and self-peptide complex presentation; in otherwords, they should not come from diseased patients. Those phenotypes areeasily monitored prior to treatment to determine the suitability of thedonor cells in this therapy. For example, conformationally specific MHCclass I and self-peptide antibodies may be used to show that thecomplexes are properly filled. In addition, we know that, in this aspectof the treatment, an increased number of matches to the HLA class Ialleles of the host results in an increase in the duration of thereversal of the disease. Desirably, at least two, and desirably all fourHLA class I alleles (e.g., the HLA A and HLA B alleles) from the donorcells are matched. Accordingly, these donor cells may be perfectlymatched or they may be semi-allogeneic (i.e., with only partial matcheson individual cells).

Treatment may involve intravenous biweekly infusions of 1×10⁷ cells ofany given donor of any given class I haplotype. It is desirable for theadministered cells to be freshly isolated and not processed withpreservatives or frozen. Cells that may be used in the methods of theinvention may be obtained, for example, from a bloodbank. In addition,semi-allogeneic cells may be obtained from a close relative of thepatient, such as a parent or a sibling. Furthermore, it would beadvantageous to have the red blood cells eliminated from thepreparations to decrease the volume of blood and lymphocytesadministered. We also determined that semi-allogeneic orfully-allogeneic irradiated cells may be used in this therapy, but theuse of irradiated cells results in a longer time course for correction.

As an alternative to administering MHC class I and peptide, anotheragent that inactivates or kills naïve T-cells can be administered.Exemplary agents include antibodies that bind and inactivate the T-cellreceptor on naïve T-cells or by binding and triggering the selectivedeath of only pathologic cells. In some embodiments, the antibodiesinhibit the activity of or naïve T-cells by at least 2, 5, 10, or15-fold more than they inhibit the activity of memory T-cells.

Simultaneously with the administration of donor cells, it is alsodesirable to induce endogenous TNF-alpha production either throughstimulation with Bacillus Clamette-Guerin (BCG) or other immuneadjuvants such as CFA, or by the direct administration of TNF-alpha. Forexample, one may administer BCG at least biweekly or, desirably, threetimes a week. Again, one skilled in the art can determine individuallythe dosing of the cells and TNF-alpha or BCG by analyzing a blood sampletwice a week for evidence of the elimination of the phenotype of thepathogenic cell. For instance, to determine the correct dose of donorMHC class I expressing cells, we may look for the elimination of theabundant naïve cells in the peripheral blood and to determine thecorrect dose of TNF-alpha or BCG, we may look for the elimination ofTNF-alpha in vitro sensitivity.

With regard to the second aspect of the therapy, TNF-alpha, BCG, oranother nonspecific form of immune stimulation may promote the inductionof endogenous TNF-alpha. For example, TNF-alpha may be administeredintramuscularly, intravesicularly, or intravenously. Moreover,recombinant human TNF-alpha or new drugs such as a TNF receptor 2agonist may be used. Such compounds have two effects, one is theelimination of apoptosis or death sensitive cells in the periphery whichcan be monitored, and the other is the promotion of endogenous beta cellregeneration, as well as possibly differentiation from the new donorblood. Exemplary doses of TNF-alpha that may be administered to apatient are approximately 40 μg/m² or 200 μg/m². Other exemplary dosesinclude doses between 2×10⁶ and 5×10⁶ mg daily for two doses in oneweek. Patients with an autoimmune disease may tolerate higher doses ofTNF-alpha and/or may require lower doses for treatment. As analternative to TNF-alpha, tolerance can be gained by cross-linking theTCR or by nonspecific vaccination through the same pathway (e.g., BCGvaccination). As an alternative to administering an inducer oflymphopenia (e.g., TNF-alpha) directly to a patient, the inducer oflymphopenia can be administered to blood obtained from the patient(e.g., blood obtained during electrophoresis), and the treated blood canbe re-administered to the patient. For induces of lymphopenia with ashort half-life (e.g., TNF-alpha) little, if any, functional compoundremains in the blood that is re-introduced into the patient. Thus, thismethod should decrease the incidence or severity of any potentialadverse, side-effects of the compound.

Any combination therapy described herein, e.g. a therapy which uses MHCclass I expressing cells and TNF-alpha induction, may be administereduntil the disease is successfully treated. For example, this therapy maybe continued for approximately 40 days; however, this time-period mayreadily be adjusted based on the observed phenotypes. Additionally, thedose of TNF-alpha can be adjusted based on the percentage of cells inblood samples from the patient that have increased sensitivity toTNF-alpha, indicating the amount of remaining autoimmune cells. Inaddition, in treating type I diabetes, it may be desirable that thepatient maintains as close to normoglycemia as possible. The murine datahave demonstrated that marked fluctuation in blood sugars hamper thenormal regenerative potential of the pancreas. Therefore, these patientsmay be placed on an insulin pump for not only the exemplary 40 days ofdisease reversing therapy, but also for a 120 day period to optimize theregenerative process. The pancreas of long-term diabetics (i.e., oneshaving diabetes for more than 15 years) may have the regenerativepotential of the pancreas diminished to such a degree that the precursorcells are no longer present. In these patients, the therapy may beidentical except for the length of the treatment. For instance, thedonor blood or bone marrow cells have to be alive for these cells toconvert to the correct tissue type, such as into beta cells of thepancreas.

As is mentioned above, some embodiments of the invention employmesodermal cells, which can be isolated from a normal donor (e.g., fromthe bone marrow, the spleen, or the peripheral blood). Typically, thiscell expresses, to a detectable degree, CD90⁺, CD44⁺, or CD29⁺, but doesnot express appreciable amounts of CD45 or CD34. This normal donor cellis administered to a person, preferably intravenously orintraperitoneally, to allow for rapid transport to the site ofinflammation, injury, or disease. Desirably, this cell is administeredto a person with active autoimmunity. Alternatively, the cell may beadministered to a person without autoimmunity or to a person withquiescent autoimmunity. The absence of active autoimmunity in a person(host) may require pretreatment of the host to initiate an inflammatoryresponse or injury at the regenerative site. In addition, pretreatmentof the donor cell may also be required. The host may be treated withTNF-, IFN-, IL-2, VEGF, FGF, or IGF-1 to prepare the blood vesselendothelium for optimal interactions with the mobilized mesodermal cell.Additionally, the pathway of VEGF-stimulated expression on endothelialcells can be enhanced with a selective inhibitor of PI-3′-kinase.Alternatively, the host can be pretreated with platelet-derived growthfactor derived from mural cells (e.g., from the neural crest orepicardium) for optimal interactions with the mobilized mesodermal cell.Additionally, the mesodermal cell can be pretreated to optimizeadherence to the endothelium. This type of therapy is envisioned to bebeneficial for the regeneration of diverse organs or organelles,including brain, skin, islets of Langerhans, heart, lung, liver, muscle,intestine, pancreas, bone, cartilage, and fat.

It may also be possible to optimize the fresh mesenchymal cell prior toinjection into the host. This can be accomplished with TNF-exposure,IL-1 exposure, or other chemical/drug treatments to increase neogenesis.

For patients that have organ or tissue damage, but no underlyingautoimmunity, it may be beneficial to avoid prolonged administration ofan immune adjuvant, e.g., TNF-alpha, as such agents may result in thedepletion of stem cells. Instead, desirably, one may induce transientlymphopenia with TNF-alpha or any other nonspecific reagent, remove thisreagent, and add cells (e.g., stem cells) to regenerate the organ ortissue. In addition, the added stem or precursor cells may be altered tohave reduced TNF-alpha sensitivity or may have increased proteasomeactivity or decreased death sensitivity through TNF or Fas. Furthermore,the host may be preconditioned with an agent that increases LMP2, LMP7,or proteasome activity (e.g., gamma interferon) prior to, concurrentwith, or after the administration of stem cells. Compounds that increaseFlt, Flk, VEGF expression or activity, hypoxia, GATA-2, hypoglycemia,IL-1, or inhibition of cAMP can also be used. Moreover, sinceadministration of TNF-alpha results in cell death due to theupregulation of Fas or FasL, it may be beneficial to precondition a hostwith an inhibitor of Fas/FasL expression or function during TNF-alpha orother immune adjuvant therapy in both patients with and withoutunderlying autoimmunity.

In contrast, administration of TNF-alpha during treatment of autoimmuneconditions typically increases the number of stem cells and thus doesnot require steps to inhibit destruction of stem cells or to replacestem cells. TNF-alpha does not deplete stem cells is in NOD mice becausemany of the stem cells in these mice have intrinsic defects in Fas andFasL expression. In contrast to normal cells, which may die due toFas/FasL upregulation that is induced by TNF-alpha, NOD stem cellssurvive. In a variety of human autoimmune diseases, the Fas/FasLdownregulation enables these human cells to survive, or even expand, inthe presence of TNF-alpha.

In a host with autoimmune disease, the signaling pathways are derangedand the administration of cytokines may have multiple effects. First,administered cytokines induce apoptosis of a subpopulation of pathologiclymphoid cells due to intrinsic errors in apoptosis resistance, thusidentifying these cells as pathogenic. Furthermore, the introduced andendogenous cytokines also promote the regeneration process presumably onthe target organ. Furthermore, if the target organ has inflammation andis exposed to the administered cytokines or processes endogenous errorsin the overexpression of proteasome function (e.g., LMP2/7 subunitexpression, a gamma responsive gene, or a TNF responsive gene), theorgan regeneration will be promoted. While not meant to limit theinvention to a particular theory, a possible mechanism of in situregeneration is that activation of the proteasome is critical for theaction of VEGF, and VEGF action is critical for Flk activity.Endothelial cells may promote this process and, with activation of theproteasome, VEGF action is accelerated thus allowing augmented Flkaction. Exogenously added stem cells may exponentially promote thisprocess, e.g., by independent proliferation or fusion with the cells orby differentiation to lineage-specific cell types. Therefore, to promotein situ organ regeneration, proteasome inhibitors are desirably avoided.A spontaneous autoimmune host in which target organ hyperexperession ofLMP2 and/or LMP7 is frequent may also have accelerated organregeneration. Organ regeneration can also be accelerated by promotingLMP2/7 hyperexpression with, e.g., gamma interferon, TNF, or a compoundthat activates the promoters of these genes, e.g., Stat1, agonists ofthe ICS-2/GAS elements in the LMP2 promoter, interferon regulatoryfactor 1(IRF1), TNF-alpha, or NFkB promoters.

Conversely, the administration of proteasome inhibitors may serve as atreatment for proliferative diseases. That is, a proteasome inhibitorcan be administered that affects an autoimmune response forproliferative cells, such as, for example, cancer cells, whilegenerating a relatively diminished autoimmune response for normal cells.Most desirably, the anti-proliferative proteasome inhibitor generates noautoimmunity to normal cells upon administration. In an additionalexample, anti-autoimmune therapy can be administered concurrent orsubsequent to the administration of proteasome inhibitors. Otherdiseases that can be treated by proteasome inhibitors include acuteinflammatory processes, such as, for example, sepsis or atherosclerosis.

Vascular endothelial growth factor (VEGF) is a potent angiogenic proteinthat enhances vascular permeability and promotes endothelial cellproliferation. VEGF stimulate two types of tyrosine kinase receptors,namely, the fms-like tyrosine kinase-1 (Flt-1) and the fetal liverkinase-1/kinase domain region (Flk-1/KDR). FGF (fibroblast growthfactor), TNF, and highly confluent cell culture induce Flk-1/FDRexpression in cells, whereas transforming growth factor 1 (TGF-1)reduces it. Thus, to promote regeneration, FGF and TNF are used, and TGFis desirably avoided. For regeneration in a normal host, the donor cellsare desirably not exposed to TNF-like substances too early because thesesubstances may accelerated death. In contrast, the host tissue may beexposed to TNF like substances or inducers of NF B or VEGF to increaseFlk-1-like expression or signaling to promote the regeneration processand/or interactions that promote in situ regeneration. Therefore, normaldonor cells may be pretreated prior to transfer to prevent death whenexposed to endogenous TNF like substances. Alternatively, the host maybe reconditioned with TNF-like substances (e.g., TNF, VEGF, FGF, or andNF B stimulator) prior to cell transfer to create an environment foroptimal proliferation. As noted above, VEGF action is dependent upon aproteasome expressing LMP2, and thus agents that induce proteasomefunction are beneficial for regeneration. One such agent is INF(interferon), which upregulates the obligatory inducible proteasomesubunits (e.g., LMP2) for optimal VEGF action.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adapt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication,patent, or patent application was specifically and individuallyindicated to be incorporated by reference.

What is claimed is:
 1. A method for treating or stabilizing anautoimmune disorder selected from the group consisting of type Idiabetes, celiac sprue-dermatitis, Crohn's disease, Graves' disease,hypothyroidism, lupus, multiple sclerosis, psoriasis, rheumatoidarthritis, sarcoidosis, Sjogren's syndrome, and ulcerative colitis in amammal comprising administering to said mammal a composition comprisinga tumor necrosis factor (TNF)-alpha receptor agonist that specificallybinds or activates TNF-alpha receptor II but not TNF-alpha receptor I.2. The method of claim 1, wherein said mammal is a human.
 3. The methodof claim 1, wherein said TNF-alpha receptor agonist treats or stabilizessaid autoimmune disorder by selectively killing blood cells withincreased sensitivity to cell death, and wherein killing of said bloodcells treats or stabilizes said disorder.
 4. The method of claim 1,wherein said TNF-alpha receptor agonist treats or stabilizes saidautoimmune disorder by promoting cellular regeneration, wherein saidregeneration treats or stabilizes said disorder.
 5. The method of claim2, wherein said disorder is type I diabetes.
 6. The method of claim 2,wherein said disorder is celiac sprue-dermatitis.
 7. The method of claim2, wherein said disorder is Crohn's disease.
 8. The method of claim 2,wherein said disorder is Graves' disease.
 9. The method of claim 2,wherein said disorder is hypothyroidism.
 10. The method of claim 2,wherein said disorder is lupus.
 11. The method of claim 2, wherein saiddisorder is multiple sclerosis.
 12. The method of claim 2, wherein saiddisorder is psoriasis.
 13. The method of claim 2, wherein said disorderis rheumatoid arthritis.
 14. The method of claim 2, wherein saiddisorder is sarcoidosis.
 15. The method of claim 2, wherein saiddisorder is Sjogren's syndrome.
 16. The method of claim 2, wherein saiddisorder is ulcerative colitis.
 17. The method of claim 3, wherein saidblood cells are T-cells, B-cells, or macrophages.
 18. The method ofclaim 1, wherein said mammal already has symptoms of said disorder. 19.The method of claim 1, wherein said TNF-alpha receptor agonist isformulated for intramuscular, intravenous, intraperitoneal,intravesicular, intraarticular, intralesional, or subcutaneousadministration.
 20. The method of claim 1, wherein said TNF-alphareceptor agonist is administered in a single dose.
 21. The method ofclaim 1, wherein said TNF-alpha receptor agonist is administered inmultiple doses.
 22. The method of claim 4, wherein said cellularregeneration comprises regeneration of an organ or tissue that isinjured, damaged, or deficient in said mammal, wherein said organ ortissue is, or is part of, bladder, brain, nervous system tissue, bloodvessels, skin, eye structures, gut, bone, muscle, ligament, cartilage,esophagus, heart, pancreas, intestines, gallbladder, bile duct, kidney,liver, lung, fallopian tubes, ovaries, prostate, spinal cord, spleen,stomach, testes, thymus, thyroid, trachea, ureter, urethra, uterus, orfat.